Sugar modified oligonucleotides that detect and modulate gene expression

ABSTRACT

Compositions and methods are provided for the treatment and diagnosis of diseases amenable to modulation of the production of selected proteins. In accordance with preferred embodiments, oligonucleotides and oligonucleotide analogs are provided which are specifically hybridizable with a selected sequence of RNA or DNA wherein at least two of the 2&#39;-deoxyfuranosyl moieties of the nucleoside unit is modified. Treatment of HIV, herpes virus, papillomavirus and other infections is provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.566,977, filed Aug. 13, 1990, now abandoned, and application Ser. No.PCT US91/00243, filed Jan. 11, 1991, that in turn is acontinuation-in-part of applications Ser. Nos. 463,358, filed Jan. 11,1990, now abandoned, and the above referenced Ser. No. 566,977, filedAug. 13, 1990, now abandoned. The entire disclosure of each of these areassigned to the assignee of this application, are incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to the design, synthesis and application ofnuclease resistant oligonucleotides which are useful for antisenseoligonucleotide therapeutics, diagnostics, and research reagents. Sugarmodified oligonucleotide which are resistant to nuclease degradation andare capable of modulating the activity of DNA and RNA are provided.Methods for modulating the production of proteins utilizing the modifiedoligonucleotide of the invention are also provided.

BACKGROUND OF THE INVENTION

It is well known that most of the bodily states in mammals includinginfectious disease states, are affected by proteins. Such proteins,either acting directly or through their enzymatic functions, contributein major proportion to many diseases in animals and man.

Classical therapeutics has generally focused upon interactions with suchproteins in efforts to moderate their disease causing or diseasepotentiating functions. Recently however, attempts have been made tomoderate the actual production of such proteins by interactions withmolecules that direct their synthesis, intracellular RNA. By interferingwith the production of proteins, it has been hoped to effect therapeuticresults with maximum effect and minimal side effects. One approach forinhibiting specific gene expression is the use of oligonucleotide andoligonucleotide analogs as antisense agents.

Antisense methodology is the complementary hybridization of relativelyshort oligonucleotides to single-stranded mRNA or single-stranded DNAsuch that the normal, essential functions of these intracellular nucleicacids are disrupted. Hybridization is the sequence specific hydrogenbonding of oligonucleotides to Watson-Crick base pairs of RNA orsingle-stranded DNA. Such base pairs are said to be complementary to oneanother.

The naturally occurring event that provides the disruption of thenucleic acid function, discussed by Cohen in Oligonucleotides: AntisenseInhibitors of Gene Expression, CRC Press, Inc., Boca Raton, Fla. (1989)is thought to be of two types. The first, hybridization arrest, denotesthe terminating event in which the oligonucleotide inhibitor binds tothe target nucleic acid and thus prevents, by simple steric hindrance,the binding of essential proteins, most often ribosomes, to the nucleicacid. Methyl phosphonate oligonucleotides; P. S. Miller & P. O. P. Ts'O,Anti-Cancer Drug Design, 2:117-128 (1987), and α-anomer oligonucleotidesare the two most extensively studied antisense agents which are thoughtto disrupt nucleic acid function by hybridization arrest.

The second type of terminating event for antisense oligonucleotidesinvolves the enzymatic cleavage of the targeted RNA by intracellularRNase H. The oligonucleotide or oligonucleotide analog, which must be ofthe deoxyribo type, hybridizes with the targeted RNA and this duplexactivates the RNase H enzyme to cleave the RNA strand, thus destroyingthe normal function of the RNA. Phosphorothioate oligonucleotides arethe most prominent example of an antisense agent which operates by thistype of antisense terminating event.

Considerable research is being directed to the application ofoligonucleotide and oligonucleotide analogs as antisense agents fortherapeutic purposes. All applications of oligonucleotides asdiagnostic, research reagents, and potential therapeutic agents requirethat the oligonucleotides or oligonucleotide analogs be synthesized inlarge quantities, be transported across cell membranes or taken up bycells, appropriately hybridize to targeted RNA or DNA, and subsequentlyterminate or disrupt nucleic acid function. These critical functionsdepend on the initial stability of oligonucleotides toward nucleasedegradation.

A serious deficiency of oligonucleotides for these purposes,particularly antisense therapeutics, is the enzymatic degradation of theadministered oligonucleotide by a variety of ubiquitous nucleolyticenzymes, intracellularly and extracellularly located, hereinafterreferred to as "nucleases". It is unlikely that unmodified, "wild type",oligonucleotides will be useful therapeutic agents because they arerapidly degraded by nucleases. Modification of oligonucleotides torender them resistant to nucleases is therefore currently a primaryfocus of antisense research.

Modifications of oligonucleotides to enhance nuclease resistance haveheretofore exclusively taken place on the sugar-phosphate backbone,particularly on the phosphorus atom. Phosphorothioates, methylphosphonates, phosphorimidates, and phosphorotriesters (phosphatemethylated DNA) have been reported to have various levels of resistanceto nucleases. However, while the ability of an antisense oligonucleotideto bind to specific DNA or RNA with fidelity is fundamental to antisensemethodology, modified phosphorous oligonucleotides, while providingvarious degrees of nuclease resistance, suffer from inferiorhybridization properties.

Due to the prochiral nature of the phosphorous atom, modifications onthe internal phosphorus atoms of modified phosphorous oligonucleotidesresult in Rp and Sp stereoisomers. Since a practical synthesis of stereoregular oligonucleotides (all Rp or Sp phosphate linkages) is unknown,oligonucleotides with modified phosphorus atoms have n² isomers with nequal to the length or the number of the bases in the oligonucleotide.Furthermore, modifications on the phosphorus atom have unnatural bulkabout the phosphorodiester linkage which interferes with theconformation of the sugar-phosphate backbone and consequently, thestability of the duplex. The effects of phosphorus atom modificationscause inferior hybridization to the targeted nucleic acids relative tothe unmodified oligonucleotide hybridizing to the same target.

The relative ability Of an oligonucleotide to bind to complementarynucleic acids is compared by determining the melting temperature of aparticular hybridization complex. The melting temperature (T_(m)), acharacteristic physical property of double helixes, denotes thetemperature in degrees centigrade at which 50% helical versus coil(unhybridized) forms are present. T_(m) is measured by using the UVspectrum to determine the formation and breakdown (melting) ofhybridization. Base stacking, which occurs during hybridization, isaccompanied by a reduction in UV absorption (hypochromicity).Consequently a reduction in UV absorption indicates a higher T_(m). Thehigher the T_(m), the greater the strength of the binding of thestrands. Non-Watson-Crick base pairing has a strong destabilizing effecton the T_(m). Consequently, absolute fidelity of base pairing isnecessary to have optimal binding of an antisense oligonucleotide to itstargeted RNA.

Considerable reduction in the hybridization properties of methylphosphonates and phosphorothioates has been reported by Cohen. Methylphosphonates have a further disadvantage in that the duplex formed withRNA does not activate degradation by RNase H as an terminating event,but instead acts by hybridization arrest which can be reversed due to ahelical melting activity located on the ribosome. Phosphorothioates arehighly resistant to most nucleases. However, phosphorothioates typicallyexhibit non-antisense modes of action, particularly the inhibition ofvarious enzyme functions due to nonspecific binding. Enzyme inhibitionby sequence-specific oligonucleotides undermines the very basis ofantisense chemotherapy.

Therefore, oligonucleotides modified to exhibit resistance to nucleases,to activate the RNase H terminating event, and to hybridize withappropriate strength and fidelity to its targeted RNA (or DNA) aregreatly desired for antisense oligonucleotide therapeutics.

M. Ikehara et al., European Journal of Biochemistry 139:447-450(1984)report the synthesis of a mixed octamer containing one2'-deoxy-2'-fluoroguanosine residue or one 2'-deoxy-2'-fluoroadenineresidue. W. Guschlbauer and K. Jankowski, Nucleic Acids Res. 8:1421(1980) have shown that the contribution of the N form (3'-endo, 2'-exo)increases with the electronegativeness of the 2'-substituent. Thus,2'-deoxy-2'-fluorouridine contains 85% of the C3'-endo conformer. M.Ikehara et al., Tetrahedron Letters 42:4073 (1979) have shown that alinear relationship between the electronegativeness of 2'-substituentsand the % N conformation (3'-endo-2'-exo) of a series of2'-deoxy-adenosines. M. Ikehara et al., Nucleic Acids Research 5:1877(1978) have chemically transformed 2'-deoxy-2'-fluoro-adenosine to its5'-diphosphate. This was subsequently enzymatically polymerized toprovide poly(2'-deoxy-2'-fluoroadenylic acid).

Furthermore, evidence was presented which indicates that 2'-substituted2'-deoxyadenosines polynucleotides resemble double stranded RNA rather,than DNA. M. Ikehara et al., Nucleic Acids Res. 5:3315 (1978) show thata 2'-fluorine substituent in poly A, poly I, and poly C duplexed totheir U, C, or I complement are significantly more stable than the riboor deoxy poly duplexes as determined by standard melting assays. M.Ikehara et al., Nucleic Acids Res. 4:4249 (1978) show that a 2'-chloroor bromo substituents in poly(2'-deoxyadenylic acid) provides nucleaseresistance. F. Eckstein et al., Biochemistry 11:4336 (1972) show thatpoly(2'-chloro-2'-deoxyuridylic acid) andpoly(2'-chloro-2'-deoxycytidylic acid) are resistant to variousnucleases. H. Inoue et al., Nucleic Acids Research 15:6131 (1987)describe the synthesis of mixed oligonucleotide sequences containing2'-OMe at every nucleotide unit. The mixed 2'-OMe substituted sequenceshybridized to their ribooligonucleotide complement (RNA) as strongly asthe ribo-ribo duplex (RNA-RNA) which is significantly stronger than thesame sequence ribo-deoxyribo heteroduplex (T_(m) s, 49.0 and 50.1 versus33.0 degrees for nonamers). S. Shibahara et al., Nucleic Acids Research17:239 (1987) describe the synthesis of mixed oligonucleotides sequencescontaining 2'-OMe at every nucleotide unit. The mixed 2'-OMe substitutedsequences were designed to inhibit HIV replication.

It is thought that the composite of the hydroxyl group's steric effect,its hydrogen bonding capabilities, and its electronegativeness versusthe properties of the hydrogen atom is responsible for the grossstructural difference between RNA and DNA. Thermal melting studiesindicate that the order of duplex stability (hybridization) of2'-methoxy oligonucleotides is in the order of RNA-RNA, RNA-DNA,DNA-DNA.

The 2'-deoxy-2'-halo, azido, amino, methoxy homopolymers of severalnatural occurring nucleosides have been prepared by polymeraseprocesses. The required 2'-modified nucleosides monomers have not beenincorporated into oligonucleotides via nucleic acids synthesizermachines. Thus, mixed sequence (sequence-specific) oligonucleotidescontaining 2'-modifications at each sugar are not known except for2'-deoxy-2'-methoxy analogs.

OBJECTS OF THE INVENTION

It is a principal object of the invention to provide nuclease resistant,sugar modified oligonucleotides or oligonucleotide analogs for use inantisense oligonucleotide diagnostics, research reagents, andtherapeutics.

It is a further object of the invention to provide such oligonucleotidesor oligonucleotides analogs which are effective in modulating theactivity of a DNA or an RNA.

Another object of the invention is to provide such oligonucleotides oroligonucleotide analogs which are less likely to invoke undesired ortoxic side reactions.

Yet another object of the invention is to provide research anddiagnostic methods and materials for assaying bodily states in animals,especially diseased states.

A further object of the invention is to provide therapeutic and researchmethods and materials for the treatment of diseases through modulationof the activity of DNA and RNA.

These and other objects will become apparent to persons of ordinaryskill in the art from a review of the present specification andattendant claims.

SUMMARY OF THE INVENTION

In accordance with the present invention, compositions which areresistant to nuclease degradation and but which modulate the activity ofDNA and RNA are provided. These compositions are comprised of sugarmodified oligonucleotides or oligonucleotide analogs, the targetingportions of which are specifically hybridizable with preselectednucleotide sequences of single-stranded or double-stranded DNA or RNA.The sugar modified oligonucleotides recognize and form double strandswith single stranded DNA and RNA or triple strands with double strandedDNA and RNA.

The nuclease resistant oligonucleotides of this invention consist of asingle strand of nucleic acid bases linked together through linkinggroups. The target portion of the nuclease resistant oligonucleotide mayrange in length from about 5 to about 50 nucleic acid bases. However, inaccordance with the preferred embodiment of this invention, a targetsequence of about 15 bases in length is optimal.

The nucleic acid bases may be pyrimidines such as thymine, uracil orcytosine, or purines such as guanine or adenine, or both, arranged in aspecific sequence. The sugar moiety of such bases may be of thedeoxyribose or ribose type. The groups linking the bases together may bethe usual sugar phosphate nucleic acid backbone, but may also bemodified as a phosphorothioate, methylphosphonate, or phosphatealkylated moiety to further enhance the sugar modified oligonucleotideproperties, along with removal of a 5'-methylene group and/orcarbocyclic sugar.

In accordance with this invention, the targeting portion is an analog ofan oligonucleotide wherein at least one of the 2'-deoxy ribofuranosylmoieties of the nucleoside unit is modified. A hydrogen or a hydroxyl,halo, azido, amino, methoxy or alkyl group may be added. For example, H,OH, F, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl, SO-alkyl, SO₂ -alkyl, ONO₂,NO₂, N₃, NH₂, NH-alkyl, OCH₂ CH═CH₂ (allyloxy), OCH═CH₂, OCCH wherealkyl is a straight or branched chain of C1 to C12 may be used, withunsaturation within the carbon chain, such as allyloxy beingparticularly preferred.

The resulting novel oligonucleotides or oligonucleotide analogs areresistant to nuclease degradation and exhibit hybridization propertiesof higher quality relative to wild type (DNA-DNA and RNA-DNA) duplexesand the phosphorus modified oligonucleotide antisense duplexescontaining phosphorothioates, methylphosphonates, phophoramidates andphosphorotriesters.

The invention is also directed to methods for modulating the productionof a protein by an organism comprising contacting the organism with acomposition formulated in accordance with the foregoing considerations.It is preferred that the RNA or DNA portion which is to be modulated bepreselected to comprise that portion of DNA or RNA which codes for theprotein whose formation is to be modulated. The targeting portion of thecomposition to be employed is, thus, selected to be complementary to thepreselected portion of DNA or RNA, that is to be an antisenseoligonucleotide for that portion.

This invention is also directed to methods of treating an organismhaving a disease characterized by the undesired production of a protein.This method comprises contacting the organism with a composition inaccordance with the foregoing considerations. The composition ispreferably one which is designed to specifically bind with messenger RNAwhich codes for the protein whose production is to be inhibited.

The invention further is directed to diagnostic methods for detectingthe presence or absence of abnormal RNA molecules or abnormal orinappropriate expression of normal RNA molecules in organisms or cells.

The invention is also directed to methods for the selective binding ofRNA for research and diagnostic purposes. Such selective, strong bindingis accomplished by interacting such RNA or DNA with compositions of theinvention which are resistant to degradative nucleases and hybridizestronger and with greater fidelity than any other known oligonucleotideor oligonucleotide analog.

Additionally this invention is directed to a method of synthesis of2'-deoxy-2'-substituted nucleosides, particularly guanosine compounds.In accordance with this method, the 2'-hydroxyl moiety of guanosine isfirst oxidized and then reduced with inversion about the 2' position toyield 9-(β-D-arabinofuranosyl)guanine. The 2' arabino hydroxyl group isderivatized with a leaving group. Nucleophilic displacement of theleaving group with a nucleophile is accomplished with a furtherinversion to give the 2'-deoxy-2'-substituted guanosine compound.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The compositions useful for modulating the activity of an RNA or DNAmolecule in accordance with this invention generally comprise a sugarmodified oligonucleotide containing a target sequence which isspecifically hybridizable with a preselected nucleotide sequence ofsingle stranded or double stranded DNA or RNA molecule and which isnuclease resistant.

It is generally desirable to select a sequence of DNA of RNA for orwhich is involved in the production of proteins whose synthesis isultimately to be modulated or inhibited in entirety. The targetingportion of the composition is generally an oligonucleotide analog. It issynthesized, conveniently through solid state synthesis of knownmethodology, to be complementary to or at least to be specificallyhybridizable with the preselected nucleotide sequence of the RNA or DNA.Nucleic acid synthesizers are commercially available and their use isgenerally understood by persons of ordinary skill in the art as beingeffective in generating nearly any oligonucleotide of reasonable lengthwhich may be desired.

In the context of this invention, the term "oligonucleotide" refers to aplurality of joined nucleotide units formed in a specific sequence fromnaturally occurring bases and pentofuranosyl groups joined through asugar group by native phosphodiester bonds. These nucleotide units maybe nucleic acid bases such as guanine, adenine, cytosine, thymine oruracil. The sugar group may be deoxyribose or ribose. This term refersto both naturally occurring or synthetic species formed from naturallyoccurring subunits.

"Oligonucleotide analog" as the term is used in connection with thisinvention, refers to moieties which function similarly tooligonucleotides but which have non-naturally occurring portions.Oligonucleotide analogs may have altered sugar moieties or inter-sugarlinkages, for example, phosphorothioates and other sulfur containingspecies which are known for use in the art. Oligonucleotide analogs mayalso comprise altered base units or other modifications consistent withthe spirit of this invention, and in particular such modifications asmay increase nuclease resistance of the oligonucleotide composition inorder to facilitate antisense therapeutic, diagnostic or researchreagent use of a particular oligonucleotide.

It is generally preferred for use in some embodiments of this inventionthat some positions of the nucleotide base be substituted in order toincrease the nuclease resistance of the composition while maintainingthe integrity of the oligonucleotide binding capabilities.

It is preferred in some embodiments of the present invention to employfurther modified oligonucleotides. In this context, modifiedoligonucleotide analogs refers to a structure which is generally similarto native oligonucleotides, but which have been modified in one or moresignificant ways.

Such modifications may take place at the sugar backbone of theinvention. It is generally preferred to enhance the ability of thetarget sequence of the sugar modified oligonucleotides to penetrate intothe intracellular spaces of cells where the messenger RNA or DNA, whichare the targets of the overall composition, reside. Therefore, it isgenerally preferred to provide modifications of oligonucleotides whichare substantially less ionic than native forms in order to facilitatepenetration of the oligonucleotide into the intracellular spaces. Any ofthe existing or yet to be discovered methods for accomplishing this goalmay be employed in accordance with the practice of the presentinvention. At present, it has been found preferable to employsubstitutions for the phosphorodiester bond, which substitutions are notonly relatively less ionic than the naturally occurring bonds but arealso substantially non-chiral.

As will be appreciated, the phosphorus atom in the phosphorodiesterlinkage is "pro-chiral". Modifications at the phosphorus, such as isdone in methyl phosphonates and phosphorothioates type oligonucleotides,results in essentially chiral structures. Chirality results in theexistence of two isomers at each chiral center which may interactdifferently with cellular molecules. Such an unresolved mixture ofisomers may inhibit the transport of the resulting compositions into theintracellular spaces or decrease the affinity and specificity ofhybridization to the specific target RNA or DNA. Thus, it is preferredin some embodiments of this invention to employ substantially non-ionic,substantially non-chiral entities in lieu of some or all of thephosphorodiester bonds. For this purpose, short chain alkyl orcycloalkyl structures especially C₂ -C₄ structures are preferred. As isset forth in an application filed on even date herewith and assigned toa common assignee hereof, said application being entitled "PolyamineOligonucleotides to Enhance Cellular Uptake," application Ser. No.558,663 filed Jul. 27, 1990 (attorney docket ISIS-24) the modificationof the sugar structure including the elimination of one of the oxygenfunctionality may permit the introduction of such substantiallynon-chiral, non-ionic substituents in this position. The entirety of thedisclosure of application Ser. No. 558,663 is incorporation herein byreference in order to disclose more fully such modifications.

In keeping with the goals of the invention are the standard backbonemodifications such as substituting P for S, Me-P, MeO-P, H₂ N-P, etc.These substitutions are thought in some cases to enhance the sugarmodified oligonucleotide properties.

The targeting portion of the compositions of the present invention, arepreferably oligonucleotide analogs having 5 to about 50 base units. Itis more preferred that such functionalities have from 8 to about 40 baseunits and even more preferred that from about 12 to 20 base units beemployed. Oligonucleotides or oligonucleotide analogs having about 15base units are preferable for the practice of certain embodiments of thepresent invention.

It is desired that the targeting portion be adapted so as to bespecifically hybridizable with the preselected nucleotide sequence ofthe RNA or DNA selected for modulation. Oligonucleotide analogsparticularly suited for the practice of one or more embodiments of thepresent invention comprise 2'-sugar modified oligonucleotides whereinone or more of the 2'-deoxy ribofuranosyl moieties of the nucleosideunit is modified with a hydrogen or hydroxyl, halo, azido, amino,alkyoxy, thioalkoxy, alkylamino or alkyl group. For example, thesubstitutions which may occur include H, OH, F, CN, CF₃, OCF₃, OCN,O-alkyl, S-alkyl, SOMe, SO₂ Me, ONO₂, NO₂, N₃, NH₂, NH-alkyl, OCH═CH₂,OCCH where alkyl is a straight or branched chain of C₁ to C₁₂ withunsaturation within the carbon chain such as allyloxy.

These modified bases are linked together and to the rest of theoligonucleotide or oligonucleotide analog through a sugar linking group.The linking group may be any of those structures described herein whichare capable of linking sugar moieties of oligonucleotides together toform the targeting portion of the compositions of this invention. It ispreferred that these sugar linking groups comprise the phosphodiesterstructure or a derivative of such. Derivatives of the phosphodiesterstructure may include substitution of a sulphur, an alkoxy group such asmethyl, methyl oxide, or amine group for an oxygen. The sugar phosphatenucleic acid backbone may be modified as a phosphorothioate,alkylphosphonate such as methylphosphonate or phosphate alkylated moiety(a phosphotriester). The phosphodiester linkage may also be replaced bya carbon or ether linkage.

In further embodiment of this invention, a linking moiety has beendevised to allow the direct attachment of a modified unit to theterminal position of the 3'-end of the modified oligonucleotides. Thus,an ester, or more preferably a bromomethylketo group, is attached to the3'-hydroxyl of a modified 2'-modified nucleoside having its 5'-hydroxylprotected with a dimethoxytriphenylmethyl group and, if the heterocycleis of the cytosine series, having that heterocycle protected with abenzoyl protecting group. If the required targeting sequence has aterminal 3'-thymine or cytosine base, the desired modified thymine orcytosine base containing the bromomethylketo linker is utilized as thefirst monomer to attach to the control pore glass (CPG) solid supportwhich contains a normal nucleoside attached via its 3'-hydroxyl group.The base sensitive ester linkage attaching the 2'-modified nucleoside tothe nucleoside attached to the CPG is cleaved under the usualconcentrated ammonium hydroxide conditions that are utilized to removethe oligonucleotide from the CPG support. This will allow the modifiedoligonucleotide to have a 2'-modified unit at its terminal, 3'-end.

Cleavage of oligonucleotides by nucleolytic enzymes require theformation of an enzyme-substrate complex, or in particular anuclease-oligonucleotide complex. The nuclease enzymes will generallyrequire specific binding sites located on the oligonucleotides forappropriate attachment. If the oligonucleotide binding sites are removedor hindered such that the nucleases will not attach to theoligonucleotides, the nuclease resistant oligonucleotides result. In thecase of restriction endonucleases that cleave sequence-specificpalindromic double-stranded DNA, certain binding sites such as the ringnitrogen in the 3- and 7-positions have been identified as requiredhiding sites. Removal of one or more of these sites or hindering thenuclease approach to these particular positions within the recognitionsequence has provided various levels or resistance to the specificnucleases.

This invention provides antisense oligonucleotides characterized bysuperior hybridizing properties. We have discovered from structureactivity relationships studies that a significant increase in binding(T_(m))s of certain 2'-sugar modified oligonucleotides to its RNA target(complement) is correlated with an increased "A" type conformation ofthe heteroduplex. Furthermore, absolute fidelity of the modifiedoligonucleotides is maintained. The increased binding of our 2'-sugarmodified sequence-specific oligonucleotides provides superior potencyand specificity compared to phosphorus modified antisenseoligonucleotides such as methyl phosphonates, phosphorothioates,phosphate triesters and phosphoramidites as known in the literature.

The only structural difference between DNA and RNA duplexes is anhydrogen atom in the 2'-position of the DNA ribofuranosyl moietiesversus a hydroxyl group in the 2'-position of the RNA ribofuranosylmoieties (assuming that the presence or absence of a methyl group in theuracil ring system has no effect). However, gross conformationaldifferences exist between DNA and RNA duplexes.

It is known from X-ray diffraction analysis of nucleic acid fibers,Arnott and Hukins, Biochemical and Biophysical Research Communication,47:1504-1510 (1970), and analysis of crystals of double-stranded nucleicacids that DNA takes a "B" form structure and that RNA only takes themuch more rigid "A" form structure. The difference between the sugarpuckering (C2' endo for "B" form DNA and C3' endo for A-form RNA) of thenucleoside monomeric units of DNA and RNA is the major conformationaldifference between double-stranded nucleic acids.

The primary contributor to the pentofuranosyl moiety conformation is thenature of the substituent in the 2'-position. Thus, the population ofthe C3'-endo form increases with respect to the C2'-endo as theelectronegativity of the 2'-substituent increases. For example, among2'-deoxy-2'-halo-adenine nucleosides, the 2'-fluoro derivative exhibitsthe largest population (65%) of C3'-endo, and the 2'-iodo shows thelowest (7%). Those of the adenosine (2'-OH) and deoxyadenosine (2'-H)are 36% and 19%, respectively. Furthermore, the effect of the 2'-fluorogroup of adenine dinucleotides(2'-deoxy-2'-fluoroadenosine-2'-deoxy-2'-fluoroadenosine or uridine) isfurther correlated to the stabilization of the stacked conformationsmore than ribo or deoxyribo modified dimers. Research indicates that thedinucleosides phosphates have a stacked conformation with a geometrysimilar to that of A--A but with a greater extent of base-baseoverlapping than A--A. It was assumed that the highly polar nature ofthe C2'-F bond and the extreme preference for C3'-endo puckering maystabilize the stacked conformation in an "A" structure.

Data from UV hypochromicity, circular dichromism, and 'H NMR alsoindicate that the degree of stacking decreases as theelectronegativeness of halogen decreases. Furthermore, a stericbulkiness in the 2'-position is better accommodated in an "A" formduplex than a "B" form duplex.

Thus, a 2'-substituent on the 3'-nucleotidyl unit of a dinucleosidemonophosphate is thought to exert a number of effects on the stackingconformation: steric repulsion, furanose puckering preference,electrostatic repulsion, hydrophobic attraction, and hydrogen bondingcapabilities. These substituent effects are thought to be determined bythe molecular size, electronegativity, and hydrophobicity of thesubstituent.

The 2'-iodo substituted nucleosides possess the lowest C3'-endopopulation (7%) of the halogen series. Thus, on steric effects alone,one would predict an 2'-iodo or similar groups would contribute stackingdestabilizing properties and thus reduced binding (T_(m))s for antisenseoligonucleotides. However, the lower electronegativeness and highhydrophobic attractive forces of the iodine atom and similar groupscomplicates the ability to predict stacking stabilities and bindingstrengths.

Studies with the 2'-OMe modification of 2'-deoxy guanosine, cytidine,and uridine dinucleoside phosphates exhibit enhanced stacking effectswith respect to the corresponding unmethylated species (2'-OH). In thiscase, the hydrophobic attractive forces of the methyl group tend toovercome the destablilizing effects of its steric bulkiness (hindrance).

2'-Fluoro-2'-deoxyadenosine has been determined to have an unusuallyhigh population of 3'-endo puckering among nucleosides. Adenosine,2'-deoxyadenosine, and other derivatives typically have population below40% in the 3'-endo conformer. It is known that a nucleoside residue inwell-stacked oligonucleotides favors 3'-endo ribofuranose puckering.

Melting temperatures (complementary binding) are increased with the2'-substituted adenosine diphosphates. It is not clear whether the3'-endo preference of the conformation or the presence of thesubstituent is responsible for the increased binding. However, as noted,greater overlap of adjacent bases (stacking) can be achieved with the3'-endo conformations.

The present novel approach to obtaining stronger binding is to prepareantisense RNA mimics to bind to the targeted RNA. Therefore, a randomstructure-activity relationship approach was undertaken to discovernuclease resistant antisense oligonucleotides that maintainedappropriate hybridization properties.

A series of 2'-deoxy-2'-modified nucleosides of adenine, guanine,cytosine, thymidine and certain analogs of these bases have beenprepared and have been inserted as the modified nucleosides intosequence-specific oligonucleotides via solid phase nucleic acidsynthesis. The novel antisense oligonucleotides were assayed for theirability to resist degradation by nucleases and to possess hybridizationproperties comparable to the unmodified parent oligonucleotide.Initially, small electronegative atoms or groups were selected becausethese type are not likely to sterically interfere with requiredWatson-Crick base pair hydrogen bonding (hybridization). However,electronic changes due to the electronegativeness of the atom or groupin the 2'-position may profoundly effect the sugar conformation. Duringour structure activity relationship studies we discovered that the sugarmodified oligonucleotides hybridized to the targeted RNA stronger thanthe unmodified (2'-deoxyribosyl type).

2'-Substituted oligonucleotides are synthesized by the standard solidphase, automated nucleic acid synthesizer such as the AppliedBiosystems, Incorporated 380B or MilliGen/Biosearch 7500 or 8800.Triester, phosphoramidite, or hydrogen phosphonate coupling chemistries(Oligonucleotides. Antisense Inhibitors of Gene Expression. M.Caruthers, pp 7-24, Edited by J. S. Cohen, CRC Press, Inc. Boca Raton,Fla., 1989) are used in with these synthesizers to provide the desiredoligonucleotides. The Beaucage reagent (Journal of American ChemicalSociety, 112, 1253-1255, 1990) or elemental sulfur (S. Beaucage et al.,Tetrahedron Letters, 22,1859-1862, 1981) is used with phosphoramidite orhydrogen phosphonate chemistries to provide 2'-substitutedphosphorothioate oligonucleotides.

The requisite 2'-substituted nucleosides (A, G, C, T(U), and nucleicacid base analogs) are generally prepared by modification of severalliterature procedures as described below.

Procedure 1. Nucleophilic Displacement of 2'-Leaving Group in ArabinoPurine Nucleosides. Nucleophilic displacement of a leaving group in the2'-up position (2'-deoxy-2'-(leaving group)arabino sugar) of adenine orguanine or their analog nucleosides. General synthetic procedures ofthis type have been described by M. Ikehara et al., Tetrahedron34:1133-1138 (1978); ibid., 31:1369-1372 (1975); Chemistry andPharmaceutical Bulletin, 26:2449-2453 (1978); ibid., 26:240-244 (1978);M. Ikehara Accounts of Chemical Research, 2:47-53 (1969); and R.Ranganathan Tetrahedron Letters, 15:1291-1294 (1977).

Procedure 2. Nucleophilic Displacement of 2,2'-Anhydro Pyrimidines.Nucleosides thymine, uracil, cytosine or their analogs are converted to2'-substituted nucleosides by the intermediacy of 2,2'-cycloanhydronucleoside as described by J. J. Fox, et al., Journal of OrganicChemistry, 29:558-564 (1964).

Procedure 3. 2'-Coupling Reactions. Appropriately 3',5'-sugar and baseprotected purine and pyrimidine nucleosides having a unprotected2'-hydroxyl group are coupled with electrophilic reagents such as methyliodide and diazomethane to provide the mixed sequences containing a2'-OMe group H. Inoue, et al., Nucleic Acids Research 15: 6131-6148.

Procedure 4. 2-Deoxy-2-substituted Ribosylations.2-Substituted-2-deoxyribosylation of the appropriately protected nucleicacid bases and nucleic acids base analogs has been reported by E. T.Jarvi, et al., Nucleosides & Nucleotides 8:1111-1114 (1989) and L. W.Hertel, et al., Journal of Organic Chemistry 53:2406-2409 (1988).

Procedure 5. Enzymatic Synthesis of 2'-Deoxy-2'-Substituted Nucleosides.The 2-Deoxy-2-substituted glycosyl transfer from one nucleoside toanother with the aid of pyrimidine and purine ribo or deoxyribophosphorolyses has been described by J. R. Rideout and T. A. Krenitsky,U.S. Pat. No. 4,381,344 (1983).

Procedure 6. Conversion of 2'-Substituents Into New Substituents.2'-Substituted-2'-deoxynucleosides are converted into new substituentsvia standard chemical manipulations. For example, S. Chladek et al.,Journal of Carbohydrates, Nucleosides & Nucleotides 7:63-75 (1980)describes the conversion of 2'-deoxy-2'-azidoadenosine, prepared fromarabinofuranosyladenine, into 2'-deoxy-2'-aminoadenosine.

Procedure 7. Free Radical Reactions. Conversions of halogen substitutednucleosides into 2'-deoxy-2'-substituted nucleosides via free radicalreactions has been described by K. E. B. Parkes and K. Taylor,Tetrahedron Letters 29:2995-2996 (1988).

Procedure 8. Conversion of Ribonucleosides to 2'-Deoxy-2'-SubstitutedNucleoside. Appropriately 3',5'-sugar and base protected purine andpyrimidine nucleosides having a unprotected 2'-hydroxyl group areconverted to 2'-deoxy-2'-substituted nucleosides by the process ofoxidation to the 2'-keto group, reaction with nucleophilic reagents, andfinally 2'-deoxygenation. Procedures of this type have been described byF. De las Heras, et al., Tetrahedron Letters 29:941-944 (1988).

Procedure 9. In a preferred process of the invention,2'-deoxy-substituted guanosine compounds are prepared via n(arabinofuranosyl)guanine intermediate obtained via anoxidation-reduction reaction. A leaving group at the 2' position of thearabinofuranosyl sugar moiety of the intermediate arabino compound isdisplaced via an SN₂ reaction with an appropriate nucleophile. Thisprocedure thus incorporate principles of both Procedure 1 and Procedure8 above. 2'-Deoxy-2'-fluoroguanosine is preferably prepared via thisprocedure. The intermediate arabino compound was obtained utilizing avariation of the oxidation-reduction procedure of Hansske, F., Madej, D.and Robins, M. J. (1984), Tetrahedron, 40:125. According to thisinvention, the reduction was effected starting at -78° C. and allowingthe reduction reaction to exothermically warm to about -2° C. Thisresults in a high yield of the intermediate arabino compound.

In conjunction with use of a low temperature reduction, utilization of atetraisopropyldisiloxane blocking group (a "TPDS" group) for the 3' and5' positions of the starting guanosine compound contributes in animproved ratio of intermediate arabino compound verses the ribo compoundfollowing oxidization and reduction. Following oxidation/reduction, theN² guanine amino nitrogen and the 2'-hydroxyl moieties of theintermediate arabino compound are blocked with isobutyryl protectinggroups ("Ibu" groups). The tetraisopropyldisiloxane blocking group isremoved and the 3' and 5' hydroxyl's are further protected with a secondblocking group, a tetrahydropyranyl blocking group (a "THP" group). Theisobutyryl group is selectively removed from 2'-hydroxyl group followedby derivation of the 2' position with a triflate (atrifluoromethylsulfonyl) leaving group. The triflate moiety was thendisplaced with inversion about the 2' position to yield the desire2'-deoxy-2'-fluoroguanosine compound.

In addition to the triflate leaving group, other leaving groups includebut are not necessarily limited to alkysulfonyl, substitutedalkylsulfonyl, arylsulfonyl, substituted arylsulfonyl,heterocyclosulfonyl or trichloroacetimidate. Representative examplesinclude p-(2,4-dinitroanilino)benzenesulfonyl, benzenesulfonyl,methylsulfonyl, p-methylbenzenesulfonyl, p-bromobenzenesulfonyl,trichloroacetimidate, acyloxy, 2,2,2-trifluoro-ethanesulfonyl,imidazolesulfonyl and 2,4,6-trichlorophenyl.

The isobutyryl group remaining on the N² heterocycllc amino moiety ofthe guanine ring can be removed to yield a completely deblockednucleoside; however, preferably for incorporation of the2'-deoxy-2'-substituted compound in an oligonucleotide, deblocking ofthe N² isobutyryl protecting group is deferred until afteroligonucleotide synthesis is complete. Normally for use on automatednucleic acid synthesizers, blocking of the N² guanine amino moiety withan isobutyryl group is preferred. Thus advantageously, the N²-isobutyryl blocked 2'-deoxy-2'-substituted guanosine compoundsresulting from the method of the invention can be directly used foroligonucleotide synthesis on automatic nucleic acid synthesizers.

The oligonucleotides or oligonucleotide analogs of this invention can beused in diagnostics, therapeutics, and as research reagents and kits.For therapeutic use the oligonucleotide is administered to an animalsuffering from a disease affected by some protein. It is preferred toadminister to patients suspected of suffering from such a disease withamounts of oligonucleotide which are effective to reduce thesymptemology of that disease. It is within the scope of a person's skillin the art to determine optimum dosages and treatment schedules for suchtreatment regimens.

It is generally preferred to apply the therapeutic agents in accordancewith this invention internally such as orally, intravenously, orintramuscularly. Other forms of administration, such as transdermally,topically, or intralesionally may also be useful. Inclusion insuppositories may also be useful. Use of pharmacologically acceptablecarriers is also preferred for some embodiments.

The following examples illustrate the practice of this invention.

EXAMPLE 1

Preparation of 2'-Deoxy-2'-fluoro Modified Oligonucleotides

A. N⁶ -Benzoyl-2'-deoxy-2'-fluoro-5'-O-(4,4'-dimethoxytrityol)!adenosine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite.

N⁶ -Benzoyl-9-(2'-fluoro-β-D-ribofuranosyl)adenine was prepared from9-β-D-arabinofuranosyladenine in a five-step synthesis using amodification of a procedure reported by M. Ikehara at al., Nucleosidesand Nucleotides 2:373-385 (1983). Thus, the N⁶ -benzoyl derivative wasobtained in good yield utilizing the method of transient protection withchlorotrimethylsilane. R. A. Jones, J. Am. Chem. Soc. 104:1316 (1982).Selective protection of the 3' and 5'-hydroxyl groups of N⁶-Benzoyl-9-β-D-arabinofuranosyladenine with tetrahydropyranyl (THP) wasaccomplished by modification of a literature procedure G. Butke, et al.,in Nucleic Acid Chemistry, Part 3:149-152, Townsend, L. B. and Tipson,R. S. eds., (J. Wiley and Sons, New York 1986) to yield N⁶ -Benzoyl-9-3',5'-di-O-(tetrahydropyran-2-yl)-β-D-arabino furanosyl!adenine in goodyield. Treatment of N⁶ -Benzoyl-9-3',5'-di-O-(tetrahydropyran-2-yl)-β-D-arabinofuranosyl! adenine withtrifluoromethanesulfonic anhydride in dichloromethane gave the2'-triflate derivative N⁶ -Benzoyl-9-2'-O-trifluoromethylsulfonyl-3',5'-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adenine which was not isolated due to its lability.Displacement of the 2'-triflate group was effected by reaction withtetrabutylammonium fluoride in tetrahydrofuran to obtain a moderateyield of the 2'-fluoro derivative N⁶ -Benzoyl-9-2'-fluoro-3',5'-di-O-tetrahydro-pyran-2-yl)-β-D-arabinofuranosyl!adenine.Deprotection of the THP groups of N⁶ -Benzoyl-9-2'-fluoro-3',5'-di-O-(tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adenine was accomplished by treatment with Dowex-50W inmethanol to yield N⁶-benzoyl-9-(2'-deoxy-2'-fluoro-β-D-ribofuranosyl)adenine in moderateyield. The ¹ H-NMR spectrum of 6 was in agreement with the literaturevalues. M. Ikehara and H. Miki, Chem. Pharm. Bull. 26: 2449-2453 (1978).Standard methodologies were employed to obtain the5'-dimethoxytrityl-3'-phosphoramidite intermediates N⁶ -Benzoyl-9-2'-fluoro-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl!adenine and N⁶-Benzoyl- 2'-deoxy-2'-fluoro-5'-O-(4,4'-dimethoxytrityl)!adenosine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite, K. K.Ogilvie, Can J. Chem. 67: 831-839 (1989).

B. N⁶ -Benzoyl-9-β-D-arabinofuranosyladenine.

9-β-D-arabinofuranosyladenine (1.07 g, 4.00 m.mol) was dissolved inanhydrous pyridine (20 mL) and anhydrous dimethylformamide (20 mL) underan argon atmosphere. The solution was cooled to ice temperature andchlorotrimethylsilane (3.88 ml, 30.6 m.mol) was added slowly to thereaction mixture via syringe. After stirring the reaction mixture at icetemperature for 30 minutes, benzoyl chloride (2.32 ml, 20 m.mol) wasadded slowly. The reaction mixture was allowed to warm to 20° C. andstirred for 2 hours. After cooling the reaction mixture to icetemperature, cold water (8 ml) was added and the mixture was stirred for15 minutes. Concentrated ammonium hydroxide (8 ml) was slowly added tothe reaction mixture to give a final concentration of 2M of ammonia.After stirring the cold reaction mixture for 30 minutes, the solvent wasevaporated in vacuo (60 torr) at 20° C. followed by evaporation in vacuo(1 torr) at 40° C. to give an oil. This oil was triturated with diethylether (50 ml) to give a solid which was filtered and washed with diethylether three times. This crude solid was triturated in methanol (100 ml)at reflux temperature three times and the solvent was evaporated toyield N⁶ -benzoyl-9-(β-D-arabinofuranosyladenine)adenine as a solid(1.50 g, 100%).

C. N⁶ -Benzoyl-9- 3",5"-di-)-tetrahydropyran-2-yl)-D-arabinofuranosyl!adenine.

N⁶ -benzoyl-9-(β-D-arabinofuranosyl)adenine (2.62 g, 7.06 m.mol) wasdissolved in anhydrous dimethylformamide (150 ml) under an argonatmosphere and p-toluenesulfonic acid monohydrate (1.32 g, 6.92 m.mol)was added. This solution was cooled to ice temperature and dihydropyran(1.26 ml, 13.8 m.mol) was added via syringe. The reaction mixture wasallowed to warm to 20° C. Over a period of 5 hours a total of 10equivalents of dihydropyran were added in 2 equivalent amounts in thefashion described. The reaction mixture was cooled to ice temperatureand saturated aqueous sodium bicarbonate was added slowly to a pH of 8,then water was added to a volume of 750 ml. The aqueous mixture wasextracted with methylene chloride four times (4×200 ml), and the organicphases were combined and dried over magnesium sulfate. The solids werefiltered and the solvent was evaporated in vacuo (60 torr) at 30° C. togive a small volume of liquid which was evaporated in vacuo (1 torr) at40° C. to give an oil. This oil was coevaporated with p-xylene in vacuoat 40° to give an oil which was dissolved in methylene chloride (100ml). Hexane (200 ml) was added to the solution and the lower-boilingsolvent was evaporated in vacuo at 30° C. to leave a white solidsuspended in hexane. This solid was filtered and washed with hexanethree times (3×10 ml) then purified by column chromatography usingsilica and methylene chloride-methanol (93:7, v/v) as eluent. The firstfraction yielded the title compound 3 as a white foam (3.19 g, 83%) anda second fraction gave a white foam (0.81 g) which was characterized asthe 5'-mono-tetrahydropyranyl derivative of N⁶-benzoyl-9-(β-D-arabinofuranosyl)adenine.

D. N⁶ -Benzoyl-9-2'-O-trifluoromethylsulfonyl-3',5'-di-O-(tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adenine.

N⁶ -Benzoyl-9-3',5'-di-O-(tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adenine (2.65 g,4.91 m.mol) was dissolved in anhydrous pyridine (20 ml) and the solventwas evaporated in vacuo (1 mm Hg) at 40° C. The resulting oil wasdissolved in anhydrous methylene chloride (130 ml) under an argonatmosphere and anhydrous pyridine (3.34 ml, 41.3 m.mol) andN,N-dimethylaminopyridine (1.95 g, 16.0 mmol) were added. The reactionmixture was cooled to ice temperature and trifluoromethanesulfonicanhydride (1.36 ml, 8.05 mmol) was added slowly via syringe. Afterstirring the reaction mixture at ice temperature for 1 h, it was pouredinto cold saturated aqueous sodium bicarbonate (140 ml). The mixture wasshaken and the organic phase was separated and kept at ice temperature.The aqueous phase was extracted with methylene chloride two more times(2×140 ml). The organic extracts which were diligently kept cold werecombined and dried over magnesium sulfate. The solvent was evaporated invacuo (60 torr) at 20° C. then evaporated in vacuo (1 torr) at 30° C. togive N⁶ -Benzoyl-9-2'-O-trifluoromethylsulfonyl-3',5'-di-O-(tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adenineas a crude oil which was not purified further.

E. N⁶ -Benzoyl-9-2'-fluoro-3',5'-di-O-(tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adenine.

N⁶ -Benzoyl-9-2'-O-trifluoromethylsulfonyl-3',5'-di-O-(tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adenine(<4.9 mmol) as a crude oil was dissolved in anhydrous tetrahydro-furan(120 ml) and this solution was cooled to ice temperature under an argonatmosphere. Tetrabutylammonium fluoride as the hydrate (12.8 g, 49.1mmol) was dissolved in anhydrous tetrahydrofuran (50 ml) and half ofthis volume was slowly added via syringe to the cold reaction mixture.After stirring at ice temperature for 1 hour, the remainder of thereagent was added slowly. The reaction mixture was stirred at icetemperature for an additional 1 hour, then the solvent was evaporated invacuo (60 torr) at 20° C. to give an oil. This oil was dissolved inmethylene chloride (250 ml) and washed with brine three times. Theorganic phase was separated and dried over magnesium sulfate. The solidswere filtered and the solvent was evaporated to give an oil. The crudeproduct was purified by column chromatography using silica in asintered-glass funnel (600 ml) and ethyl acetate was used as eluent. N⁶-Benzoyl-9-2'-deoxy-2'-fluoro-3',5'-di-O-(tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adeninewas obtained as an oil (2.03 g, 76%).

F. N⁶ -Benzoyl-9-(2'-deoxy-2'-fluoro-β-D-ribofuranosyl)adenine.

N⁶ -Benzoyl-9-2'-fluoro-3',5'-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adenine(1.31 g, 2.42 mmol) was dissolved in methanol (60 ml), and Dowex50W×2-100 (4 cm3, 2.4 m.eq) was added to the reaction mixture. Thereaction mixture was stirred at 20° C. for 1 hour then cooled to icetemperature. Triethylamine (5 ml) was then slowly added to the coldreaction mixture to a pH of 12. The resin was filtered and washed with30% triethylamine in methanol until the wash no longer contained UVabsorbing material. Toluene (50 ml) was added to the washes and thesolvent was evaporated at 24° C. in vacuo (60 torr then 1 torr) to givea residue. This residue was partially dissolved in methylene chloride(30 ml) and the solvent was transferred to a separatory funnel. Theremainder of the residue was dissolved in hot (60° C.) water and aftercooling the solvent it was also added to the separatory funnel. Thebiphasic system was extracted, and the organic phase was separated andextracted three times with water (3×100 ml). The combined aqueousextracts were evaporated in vacuo (60 torr then 1 torr Hg) at 40° C. togive an oil which was evaporated with anhydrous pyridine (50 ml). Thisoil was further dried in vacuo (1 torr Hg) at 20° C. in the presence ofphosphorous pentoxide overnight to give N⁶-benzoyl-9-(2'-deoxy-2'-fluoro-β-D-ribofuranosyl)adenine as a yellowfoam (1.08 g, 100%) which contained minor impurities.

G. N⁶ -benzoyl-9-2'-fluoro-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl!adenine.

N⁶ -benzoyl-9-(2'-fluoro-b-D-ribofuranosyl)adenine (1.08 g, 2.89 mmol)which contained minor impurities was dissolved in anhydrous pyridine (20ml) under an argon atmosphere, and dry triethylamine (0.52 ml, 3.76mmol) was added followed by addition of 4,4'-dimethoxytrityl chloride(1.13 g, 3.32 mmol). After 4 hours of stirring at 20° C. the reactionmixture was transferred to a separatory funnel and diethyl ether (40 ml)was added to give a white suspension. This mixture was washed with waterthree times (3×10 ml), the organic phase was separated and dried overmagnesium sulfate. Triethylamine (1 ml) was added to the solution andthe solvent was evaporated in vacuo (60 torr Hg) at 20° C. to give anoil which was evaporated with toluene (20 ml) containing triethylamine(1 ml). This crude product was purified by column chromatography usingsilica and ethyl-acetate-triethylamine (99:1, v/v) followed by ethylacetate-methanol-triethylamine (80:19:1) to give the product in twofractions. The fractions were evaporated in vacuo (60 torr then 1 torrHg) at 20° C. to give a foam which was further dried in vacuo (1 torrHg) at 20° C. in the presence of sodium hydroxide to give N⁶ -benzoyl-9-2'-fluoro-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl!adenine as afoam (1.02 g, 52%).

H. N⁶ -Benzoyl-2'-fluoro-5'-O-(4,4'-dimethoxytrityl)!adenosine-3'-O-N,N-diisopropyl-β-cyanoethylphosphoramidite.

N⁶ -Benzoyl-9-2'-fluoro-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl!adenine (1.26 g,1.89 mmol) was dissolved in anhydrous dichloromethane (13 ml) under anargon atmosphere, diisopropylethylamine (0.82 ml, 4.66 mmol) was added,and the reaction mixture was cooled to ice temperature.Chloro(diisopropylamino)-β-cyanoethoxyphosphine (0.88 ml, 4.03 mmol) wasadded to the reaction mixture which was allowed to warm to 20° C. andstirred for 3 hours. Ethyl acetate (80 ml) and triethylamine (1 ml) wereadded and this solution was washed with brine solution three times (3×25ml). The organic phase was separated and dried over magnesium sulfate.After filtration of the solids the solvent was evaporated in vacuo at20° C. to give an oil which was purified by column chromatography usingsilica and hexane-ethyl acetate-triethyl-amine (50:49:1) as eluent.Evaporation of the fractions in vacuo at 20° C. gave a foam which wasevaporated with anhydrous pyridine (20 ml) in vacuo (1 torr) at 26° C.and further dried in vacuo (1 torr Hg) at 20° C. in the presence ofsodium hydroxide for 24 h to give N⁶ -benzoyl-2'-deoxy-2'-fluoro-5'-O-(4,4'-dimethoxytrityol)!adenosine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramiditeas a foam (1.05 g, 63%).

I.2'-Deoxy-2'-fluoro-5'-O-(4,4'-dimethoxytrityl)-uridine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

2,2'-Cyclouridine is treated with a solution of 70% hydrogenfluoride/pyridine in dioxane at 120° C. for ten hours to provide aftersolvent removal a 75% yield of 2'-deoxy-2'-fluorouridine. The 5'-DMT and3'-cyanoethoxydiisopropylphosphoramidite derivitized nucleoside isobtained by standard literature procedures, M. J. Gait, ed.,Oligonucleotide Synthesis. A Practical Approach, (IRL Press, Washington,D.C., 1984) or through the procedure of Example 1A.

J.2'-Deoxy-2'-fluoro-5'-O-(4,4'-dimethoxytrityl)-cytidine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

2'-Deoxy-2'-fluorouridine is converted to the corresponding cytidineanalog via a triazolo intermediate that in turn was aminated theheterocycle is then protected by selective N⁴ -benzoylation. The5'-O-(4,4'-dimethoxy-trityl)-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite)can be prepared in accordance with Example 1A.

K. 9-(3',5'-1,1,3,3'-Tetraisopropyldisilox-1,3-diyl!-β-D-arabinofuranosyl)guanine.

The 3' and 5' positions of guanosine were protected by the addition of aTPDS (1,1,3,3-tetraisopropyldisilox-1,3-diyl) protecting group as perthe procedure of Robins, M. J., Wilson, J. S., Sawyer, L. and James, M.N. G. (1983) Can. J. Chem., 61:1911. To a stirred solution of DMSO (160mls) and acetic anhydride (20.0 ml, 212 mmol) was added the TPDSguanosine (21.0 g, 0.040 mol). The reaction was stirred for 36 hrs atroom temperature and then cooled to 0° C. Cold EtOH (400 ml, 95%) wasadded and the reaction mixture further cooled to -78° C. in a dryice/acetone bath. NaBH₄ (2.0 g, 1.32 mol.eq) was added. The reaction wasallowed to come to -2° C., stirred at -2° C. for 30 mins, and againcooled to -78° C. This was repeated twice more. After addition of theNaBH₄ was complete, the reaction was stirred at ice temperature for 30mins and then at RT for 1 hr. The reaction was taken up in EtOAc (11)and washed 2× with a saturated NaCl solution. The organic layer wasdried over MgSO₄ and evaporated at RT. The residue was co-evaporated 2×with toluene and purified by silica gel column chromatography using CH₂Cl₂ -MeOH (90:10) as the eluent. 6.02 g of pure product precipitate fromthe appropriate column fractions during evaporation of these fractionand an additional 11.49 g of product was obtained as a residue uponevaporated of the fractions.

L. N² -Isobutyryl-9-(2'-O-isobutyryl-3',5'-1,1,3,3-tetraisopropyldisilox-1,3-diyl!-β-D-arabinofuranosyl)guanine.

9-(3',5'-1,1,3,3-Tetraisopropyldisilox-1,3-diyl!-β-D-arabinofuranosyl)guanine(6.5 g, 0.01248 mol) was dissolved in anhydrous pyridine (156 ml) underargon. DMAP (9.15 g) was added. Isobutyric anhydride (6.12 ml) wasslowly added and the reaction mixture stirred at RT overnight. Thereaction mixture was poured into cold sat. NaHCO₃ (156 ml) and stirredfor 10 min. The aqueous solution was extracted 3× with EtOAc (156 ml).The organic phase was washed 3× with sat. NaHCO₃ and evaporated todryness at RT. The residue was co-evaporated with toluene at RT. Theresidue was purified by silica gel column chromatography using CH₂ Cl₂-acetone (85:15) to yield 5.67 g (68%) of product.

M. N² -Isobutyryl-9-(2'-O-isobutyryl-β-D-arabinofuranosyl)guanine.

N² -Isobutyryl-9-(2'-isobutyryl-3',5'-1,1,3,3-tetraisopropyldisilox-1,3-diyl!-β-D-arabinofuranosyl)guanine(9.83 g, 0.01476 mol) was dissolved in anhydrous THF (87.4 ml) at RTunder argon. 1M N(nBu)₄ F in THF (29.52 ml, 2 eq.) was added and themixture stirred for 1/2 hr. The reaction mixture was evaporated at RTand the residue purified by silica gel column chromatography usingEtOAc-MeOH (85:15) to yield 4.98 g (80%) of product.

N. N² -Isobutyryl-9-(2'-O-isobutyryl-3',5'-di-O-tetrahydropyran-2-yl!-β-D-arabinofuranosyl)guanine.

N² -Isobutyryl-9-(2'-O-isobutyryl-β-D-arabinofuranosyl)guanine (4.9 g)was dissolved in anhydrous 1,4-dioxane (98 ml) at RT under argon.p-Toluenesulfonic acid monohydrate (0.97 g, 0.44 eq.) was added followedby 3,4-dihydro-2H-pyran, i.e. DHP, (9.34 ml, 8.8 sq.). The mixture wasstirred for 2 hrs then cooled to ice temp and sat. NaHCO₃ (125 ml) wasadded to quench the reaction. The reaction mixture was extracted 3× with125 ml portions of CH₂ Cl₂ and the organic phase dried over MgSO₄. Theorganic phase was evaporated and the residue dissolved in a minimum, butsufficient amount to yield a clear liquid not a syrup, volume of CH₂ Cl₂and dripped into 100 times the CH₂ Cl₂ volume of hexane. Theprecipitated was filtered to give 5.59 g (81.5%) of product.

O. N² -Isobutyryl-9-(3',5'-di-O-tetrahydropyran-2-yl!-β-D-arabinofuranosyl)guanine.

N² -Isobutyryl-9-(2'-O-isobutyryl-3',5'-di-O-tetrahydropyran-2-yl!-β-D-arabinofuranosyl)guanine (5.58 g) wasdissolved in pyridine:MeOH:H₂ O (65:30:15, 52 ml) at RT. The solutionwas cooled to ice temp and 52 ml of 2N NaOH in EtOH-MeOH (95:15) wasadded slowly followed by stirring for 2 hrs at ice temp. Glacial AcOHwas added to pH6. Sat. NaHCO₃ was then added to pH 7. The mixture wasevaporated at RT and the residue co-evaporated with toluene. The residuewas dissolved in EtOAc (150 ml) and wash 3× with sat. NaHCO₃. Theorganic phase was evaporated and the residue purified by silica gelcolumn chromatography using EtOAc-MeOH (95:5) to yield 3.85 g (78.3%) ofproduct.

P. N² -Isobutyryl-9-(3',5'-di-O-tetrahydropyran-2-yl!-2'-O-trifluormethylsulfonyl-β-D-arabinofuranosyl)guanine.

N² -Isobutyryl-9-(3',5'-di-O-tetrahydropyran-2-yl!-β-D-arabinofuranosyl)guanine (3.84 g) wasdissolved in anhydrous CH₂ Cl₂ (79 ml), anhydrous pyridine (5.0 ml) and4-dimethylaminopyridine (2.93 g) at RT under argon. The solution wascooled to ice temp. and trifluoromethanesulfonic anhydride (1.99 ml) wasslowly added with stirring. The mixture was stirred for 1 hr then pouredinto 100 ml of sat. NaHCO₃. The aqueous phase was extracted 3× with coldCH₂ Cl₂. The organic phase was dried over MgSO₄, evaporated andco-evaporated with anhydrous CH₃ CN at RT to yield the crude product.

Q. N² -Isobutyryl-9-(2'-deoxy-2'-fluoro-3',5'-di-O-tetrahydropyran-2-yl!-β-D-ribofuranosyl)guanine.

The crude product from Example 1-P, i.e. N² -isobutyryl-9-(3',5'-di-O-tetrahydropyran-2-yl!-2'-O-trifluormethylsulfonyl-β-D-arabinofuranosyl)guanine,was dissolved in anhydrous THF (113 ml) under argon at ice temp. 1Manhydrous N(nBu)₄ F (dried by co-evaporation with pyridine) in THF(36.95 ml) was added with stirring. After 1 hr a further aliquot of 1MN(nBu)₄ F in THF (36.95 ml) (10 mol. eq. total) was added. The mixturewas stirred for 5 hrs at ice temp. and stored in a -30° C. freezerovernight. The reaction mixture was evaporated at RT and the residuedissolved in CH₂ Cl₂ (160 ml) and extracted 5× with deionized H₂ O. Theorganic phase was dried over MgSO₄ and evaporated. The residue waspurified by silica gel column chromatography using EtOAc-MeOH (95:5) toyield 5.25 g of product.

R. N² -Isobutyryl-9-(2'-deoxy-2'-fluoro-β-D-ribofuranosyl)guanine

N² -Isobutyryl-9-(2'-deoxy-2'-fluoro-3',5'-di-O-tetrahydropyran-2-yl!-β-D-ribofuranosyl)guanine (3.85 g) was dissolvedin MeOH (80 ml) at RT. 12.32 cm³ of pre-washed Dowex 50W resin was addedand the mixture stirred at RT for 1 hr. The resin was filtered and thefiltrate evaporated to dryness. The resin was washed withpyridinetriethylamino-H₂ O (1:3:3) until clear. This filtrate wasevaporated to an oil. The residues from the two filtrates were combinedin H₂ O (200 ml) and washed 3× with CH₂ Cl₂ (100 ml). The aqueous phasewas evaporated to dryness and the residue recrystallized from hot MeOHto yield a 0.299 g first crop of product as a white powder. Theremaining MeOH solution was purified by silica gel column chromatographyyielding a further crop of 0.783 g by elution with EtOH-MeOH (80:20).

S. N² -Isobutyryl-9-(2'-deoxy-2'-fluoro-5'-O-4,4'-dimethoxytrityl!-β-D-ribofuranosyl)guanine.

N² -Isobutyryl-9-(2'-deoxy-2'-fluoro-β-D-ribofuranosyl)guanine (1.09 g)was dissolved in pyridine (20 ml) and triethylamine (0.56 ml) at RTunder argon. 4,4'-Dimethoxytrityl chloride (1.20 g, 1.15 molar eq.) wasadded and the mixture stirred at RT for 5 hrs. The mixture wastransferred to a separatory funnel and extracted with Et₂ O (100 ml).The organic phase was washed 3× with sat. NaHCO₃ (70 ml portions) andthe aqueous phase back extracted 3× with Et₂ O. The combined organicphases were dried over MgSO₄ and triethylamine (4 ml) added to maintainthe solution basic. The solvent was evaporated and the residue purifiedby silica gel column chromatography. The column was eluted withEtOAc-Et₃ N (100:1) and then EtOAc-MeOH-Et₃ N (95:5:1) to yield 1.03 gof product. ¹ H-NMR (DMSO-d₆) δ 6.09 (dd, 1, H1', J₁₋₂ =2.61, J_(1'),F=16.2 Hz); δ 5.28 (ddd, 1, H2', J_(2'-F) =52.8 Hz); δ 4.38 (m, 1, H3',J_(3'),F =19.8 Hz).

T. N² -Isobutyzyl-9-(2'-deoxy-2'-fluoro-5'-O-4,4'-dimethoxytrityl!)guanosine-3'-O-N,N-diisopropyl-β-cyanoethylphosphoramidite.

N² -Isobutyryl-9-(2'-deoxy-2'-fluoro-5'-O-4,4'-dimethoxytrityl!-β-D-ribofuranosyl)guanine (0.587 g) was dissolvedin anhydrous CH₂ Cl₂ (31 ml) and diisopropylethylamine (0.4 ml) at RTunder argon. The solution was cooled to ice temp andchloro(diisopropylamino)-β-cyanoethoxyphosphine (0.42 ml) was slowlyadded. The reaction was allowed to warm to RT and stirred for 3.5 hrs.CH₂ Cl₂ -Et₃ N (100:1, 35 ml) was added and the mixture washed 1× withsat. NaHCO₃ (6 ml). The organic phase was dried over MgSO₄ andevaporated at RT. The residue was purified by silica gel columnchromatography using Hex-EtOAc-Et₃ N (75:25:1) for 2 column volumes,then Hex-EtOAc-Et₃ N (25:75:1) and finally EtOAc-Et₃ N. The productcontaining fractions were pooled and evaporated at RT. The resulting oilwas co-evaporated 2× with CH₃ CN and placed on a vacuum pump overnightto dry. The resulting white solid was dissolved in CH₂ Cl₂ (3 ml) anddripped into stirring hexane (300 ml). The resulting precipitate wasfiltered and dried on a vacuum pump to yield 0.673 g (88%) of product.³¹ P-NMR (CDCl₃) δ 150.5, 151.5.

EXAMPLE 2

Preparation of 2'-Deoxy-2'-cyano Modified Oligonucleotides

A. N⁶ -Benzoyl- 2'-deoxy-2'-cyano-5'-O-(4,4'-dimethoxytrityl)!adenosine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

2'-Deoxy-2'-cyanoadenosine is prepared by the free radical replacementof the 2'-iodo group of2'-deoxy-2'-iodo-3',5'-O-(disiloxytetraisopropyl)-N6-benzoyladenosineaccording to a similar procedure described by K. E. B. Parkes and K.Taylor, Tetrahedron Letters 29:2995-2996 (1988).2'-Deoxy-2'-iodoadenosine was prepared by R. Ranganathan as described inTetrahedron Letters 15:1291-1294 (1977), and disilyated as described byW. T. Markiewicz and M. Wiewiorowski in Nucleic Acid Chemistry, Part 3,pp. 222-231, Townsend, L. B.; Tipson, R. S. eds. (J. Wiley and Sons, NewYork, 1986). This material is treated with hexamethylditin, AIBN, andt-butylisocyanate in toluene to provide protected2'-deoxy-2'-cyanoadenosine. This material, after selective deprotection,is converted to its 5'-DMT-3'-phosphoramidite as described in Example1A.

B.2'-Deoxy-2'-cyano-5'-O-(4,4'-dimethoxytrityl)uridine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

2'-Deoxyuridine (or 5-methyluridine), 3',5'-disilylated as describedabove, is converted to the 2'-iodo derivative by triphenylphosphoniummethyl iodide treatment as described by K. E. B. Parkes and K. Taylor,Tetrahedron Letters 29:2995-2996 (1988). Application of free radicalreaction conditions as described by K. E. B. Parkes and K, Taylor,Tetrahedron Letters 29:2995-2996 (1988), provides the 2'-cyano group ofthe protected nucleoside. Deprotection of this material and subsequentconversion to the protected monomer as described above provides therequisite nucleic acid synthesizer material.

C.2'-Deoxy-2'-cyano-5'-O-(4,4'-dimethoxytrityl)cytidine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite),

2'-Deoxy-2'-iodocytidine is obtained from the corresponding abovedescribed uridine compound via a conventional keto to amino conversion.

D.2'-Deoxy-2'-cyano-5'-O-(4,4'-dimethoxytrityl)guanosine-3'-O-(N,N-diisopropyl-β-cyanoethylphos-phoramidite).

2'-Deoxy-2'-cyanoguanosine is obtained by the displacement of thetriflate group in the 2'-up position (arabino sugar) of3',5'-disilylated N2-isobutrylguanosine. Standard deprotection andsubsequent reprotection provides the title monomer.

EXAMPLE 3

Preparation of 2'-Deoxy-2'-(trifluoromethyl) Modified Oligonucleotides

The requisite 2'-deoxy-2'-trifluromethyribosides of nucleic acid basesA, G, U(T), and C are prepared by modifications of a literatureprocedure described by Q.-Y. Chen and S. W. Wu in the Journal ofChemical Society Perkin Transactions 2385-2387 (1989). Standardprocedures, as described in Example 1A, are employed to prepare the5'-DMT and 3'-phosphoramidites as listed below.

A. N⁶ -Benzoyl-2'-deoxy-2'-trifluoromethyl-5'-O-(4,4'-dimethoxytrityl)!adenosine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

B.2'-Deoxy-2'-trifluoromethyl-5'-O-(4,4'-dimethoxytrityl)uridine-3'-O-(N,N-diisopropyl-β-cyanoethyl-phosphoramidite).

C.2'-Deoxy-2'-trifluoromethyl-5'-O-(4,4'-dimethoxytrityl)cytidine-3'-O-(N,N-diisopropyl-β-cyanoethyl-phosphoramidite).

D.2'-Deoxy-2'-trifluoromethyl-5'-O-(4,4'-dimethoxytrityl)guanosine-3'-O-(N,N-diisopropyl-β-cyano-ethylphosphoramidite).

EXAMPLE 4

Preparation of 2'-Deoxy-2'-(trifluoromethoxy) Modified Oligonucleotides

The requisite 2'-deoxy-2'-O-trifluoromethylribosides of nucleic acidbases A, G, U(T), and C are prepared by modifications of literatureprocedures described by B. S. Sproat, et al., Nucleic Acids Research18:41-49 (1990) and H. Inoue, et al., Nucleic Acids Research15:6131-6148 (1987). Standard procedures, as described in Example 1A,are employed to prepare the 5'-DMT and 3'-phosphoramidites as listedbelow.

A. N6-Benzoyl-2'-deoxy-2'-(trifluoromethoxy)-5'-O-(4,4'-dimethoxytrityl)!adenosine-3'-O-(N,N-diisoporopyl-β-cyanoethylphosphoramidite).

B.2'-Deoxy-2'-(trifluoromethoxy)-5'-O-(4,4'-dimethoxytrityl)uridine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

C.2'-Deoxy-2'-(trifluoromethoxy)-5'-O-(4,4'-dimethoxytrityl)cytidine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

D.2'-Deoxy-2'-(trifluoromethoxy)-5'-O-(4,4'-dimethoxytrityl)guanosine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

EXAMPLE 5

Preparation of 2'-Deoxy-2'-(1-proproxy) Modified Oligonucleotides

The requisite 2'-deoxy-2'-O-propyl ribosides of nucleic acid bases A, G,U(T), and C are prepared by modifications of literature proceduresdescribed by B. S. Sproat, et al., Nucleic Acids Research 18:41-49(1990) and H. Inoue, et al., Nucleic Acids Research 15:6131-6148 (1987).Standard procedures, as described in Example 1A, are employed to preparethe 5'-DMT and 3'-phosphoramidites as listed below.

A. N⁶ -Benzoyl-2'-deoxy-2'-(1-proproxy)-5'-O-(4,4'-dimethoxytrityl)!adenosine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

B.2'-Deoxy-2'-(1-proproxy)-5'-O-(4,4'-dimethoxytrityl)uridine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

C.2'-Deoxy-2'-(1-proproxy)-5'-O-(4,4'-dimethoxytrityl)cytidine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

D.2'-Deoxy-2'-(1-proproxy)-5'-O-(4,4'-dimethoxytrityl)guanosine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

EXAMPLE 6

Preparation of 2'-Deoxy-2'-(vinyloxy) Modified Oligonucleotides

The requisite 2'-deoxy-2'-O-vinyl ribosides of nucleic acid bases A, G,U(T), and C are prepared by modifications of literature proceduresdescribed by B. S. Sproat, et al., Nucleic Acids Research 18:41-49(1990) and H. Inoue, et al., Nucleic Acids Research 15:6131-6148 (1987).In this case 1,2-dibromoethane is coupled to the 2'-hydroxyl andsubsequent dehydrobromination affords the desired blocked 2'-vinylnucleoside. Standard procedures, as described in Example 1A, areemployed to prepare the 5'-DMT and 3'-phosphoramidites as listed below.

A. N⁶ -Benzoyl-2'-deoxy-2'-(vinyloxy)-5'-O-(4,4'-dimethoxytrityl)!adenosine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

B.2'-Deoxy-2'-(vinyloxy)-5'-O-(4,4'-dimethoxytrityl)uridine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

C.2'-Deoxy-2'-(vinyloxy)-5'-O-(4,4'-dimethoxyltrityl)cytidine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

D.2'-Deoxy-2'-(vinyloxy)-5'-O-(4,4'-dimethoxytrityl)guanosine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

EXAMPLE 7

Preparation of 2'-Deoxy-2'-(allyloxy) Modified Oligonucleotides

The requisite 2'-deoxy-2'-O-allyl ribosides of nucleic acid bases A, G,U(T), and C are prepared by modifications of literature proceduresdescribed by B. S. Sproat, et al., Nucleic Acids Research 18:41-49(1990) and H. Inoue, et al., Nucleic Acids Research 15:6131-6148 (1987).Standard procedures, as described in Example 1A, are employed to preparethe 5'-DMT and 3'-phosphoramidites as listed below.

A. N⁶ -Benzoyl-2'-deoxy-2'-(allyloxy)-5'-(4,4'-dimethoxytrityl)!adenosine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphorsmidite).

B.2'-Deoxy-2'-(allyloxy)-5'-O-(4,4'-dimethoxytrityl)-uridine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

C.2'-Deoxy-2'-(allyloxy)-5'-O-(4,4'-dimethoxytrityl)-cytidine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

D.2'-Deoxy-2'-(allyloxy)-5'-O-(4,4'-dimethoxytrityl)-guanosine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite).

EXAMPLE 8

Preparation of 2'-Deoxy-2'-(methylthio), (methylsulfinyl) and(methylsulfonyl) Modified Oligonucleotides

A. 2'-Deoxy-2'-Methylthiouridine

2,2'-Anhydrouridine (15.5 g, 68.2 mmol) Rao, T. S. and Reese, C. B.(1989) J. Chem. Soc., Chem. Commun., 997!, methanethiol (15.7 g, 327mmol), 1,1,3,3-tetramethylguanidine (39.2 g, 341 mmol) anddimethylforamide (150 ml), were heated together at 60° C. After 12 hr,the reaction mixture was cooled and concentrated under reduced pressure.The residual oil was purified by flash column chromatography on silicagel (300 g). Concentration of the appropriate, fractions, which wereeluted with CH₂ Cl₂ -MeOH (9:1, v/v), and drying of the residue underhigh vacuum gave 2'-deoxy-2'-methylthiouridine as a pale yellow solid(14.11 g, 75.4%). Attempts to crystallize the solids fromethanol-hexanes (as reported by Imazawa, M., Ueda, T., and Ukita, T.(1975) Chem. Pharm. Bull., 23:604) failed and the material turned into ahygroscopic foam.

¹ H NMR (Me₂ SO-d₆) δ 2.0 (3H, s, SCH₃), 3.34 (1H, dd, J_(3'),2' =54 Hz,2'H), 3.59 (2H, br m, 5'CH₂), 3.84 (1H, m, 4'H), 4.2 (1H, dd, J_(3'),4'=2.2 Hz, 3'H), 5.15 (1H, t, 5'OH), 5.62 (1H, t, 3'OH), 5.64 (1H, d,J_(C6),C5 =8.2 Hz), 6.02 (1H, d, J_(1'),2' =6 Hz, 1'H), 7.82 (1H, d,J_(C5),C6 =8.2 Hz, C₆ H), 11.38 (1H, br s, NH).

B. 2,2'-Anhydro-5-Methyluridine

A mixture of 5-methyluridine (16.77 g, 69.2 mmol), diphenyl carbonate(17.8 g, 83.1 mmol) and sodium bicarbonate (100 mg) inhexamethylphosphoramide (175 ml) was heated to 150° C. with stirringuntil evolution of CO₂ ceased (approximately 1 hr). The reaction mixturewas cooled and then poured into diethylether (11) while stirring tofurnish a brown gum. Repeated washings with diethylether (4×250 ml)furnished a straw colored hygroscopic powder. The solid was purified byshort column chromatography on silica gel (400 g). Pooling andconcentration of appropriate fractions, which were eluted with CH₂ Cl₂-MeOH (85:15, v/v) furnished the title compound as a straw colored solid(12 g, 77.3%) which crystallized from EtOH as long needles, m.p.226°-227° C.

C. 2'-Deoxy-2'-Methylthio-5-Methyluridine

2.2'-Anhydro-5-methyluridine (17.02 g, 70.6 mmol), methanethiol (16.3 g,339 mmol), 1,1,3,3-tetramethylguanidine (40.6 g, 353 mmol), anddimethylformamide (150 ml) were heated together at 60° C. After 12 hr,the products were cooled and concentrated under reduced pressure. Theresidual oil was purified by short silica gel (300 g) columnchromatography. Concentration of appropriate fractions, which wereeluted with CH₂ Cl₂ -MeOH (93:7, v/v), furnished the title compound as awhite foam (15.08 g, 74.1%). Crystallization from EtOH-CH₂ Cl₂ furnishedwhite needles.

D. 2'-Deoxy-2'-Methylsulfinyluridine

To a stirred solution of 2'-deoxy-2'methylthiouridine (1 g, 3.65 mmol)in EtOH (50 ml) was added a solution of m-chloroperbenzoic acid (50%,1.26 g, 3.65 mmol in 50 ml EtOH) over a period of 45 min at 0° C. Thesolvent was removed under vacuum and the residue purified by shortsilica gel (30 g) column chromatography. Concentration of appropriatefractions, which were eluted with CH₂ Cl₂ -MeOH (75:25, v/v), affordedthe title compound as a white solid (0.65 g, 61.4%). Crystallizationfrom EtOH furnished white granules, m.p. 219°-221° C.

¹ H NMR (Me₂ SO-d₆) δ 2.5(3H, s, SOCH₃), 3.56 (2H, br s, 5'CH₂), 3.8(1H, m, 4'H), 3.91 (1H, m, 2'H), 4.57 (1H, m, 3'H), 5.2 (1H, br s,5'OH), 5.75 (1H, d, C₅ H), 6.19 (1H, d, 3'OH), 6.35 (1H, d, 1'H), 7.88(1H, d, C₆ H), 11.43 (1H, br s, NH).

E. 2'-Deoxy-2'-Methylsulfonyluridine

To a stirred solution of 2'-deoxy-2'-methyluridine (1 g, 3.65 mmol) inEtOH (50 ml) was added m-chloroperbenzoic acid (50%, 3.27 g, 14.6 mmol)in one portion at room temperature. After 2 hr., the solution wasfiltered to collect a white precipitate, which on washing (2×20 ml, EtOHand 2×20 ml Et₂ O) and drying furnished the title compound as a finepowder (0.76 g, 68%), m.p. 227°-228° C.

¹ H NMR (Me₂ SO-d₆) δ 3.1 (3H, s, SO₂ CH₃), 3.58 (2H, m, 5'CH₂), 3.95(1H, m, 2'H), 3.98 (1H, m, 4'H), 4.5 (1H, br s, 3'H), 5.2 (1H, br s,5'OH), 5.75 (1H, d, C₅ H), 6.25 (1H, d, 3'OH), 6.5 (1H, d, 1'H), 7.8(1H, d, C₆ H), 11.45 (1H, br s, NH).

F. 2'-Deoxy-5-O-(4,4'-Dimethoxytrityl)-2'-Methylthiouridine

To a stirred solution of 2'-deoxy-2'-methylthiouridine (1.09 g, 4 mmol))in dry pyridine (10 ml) was added 4,4'-dimethoxytritylchloride (1.69 g,5 mmol) and 4-dimethylaminopyridine (50mg) at room temperature. Thesolution was stirred for 12 hr and the reaction quenched by adding MeOH(1 ml). The reaction mixture was concentrated under vacuum and theresidue dissolved in CH₂ Cl₂ (100 ml), washed with sat. aq. NaHCO₃ (2×50ml), sat. aq. NaCl (2×50 ml), and dried (MgSO₄). The solution wasconcentrated under vacuum and the residue purified by silica gel (30 g)column Chromatography. Elution with CH₂ Cl₂ -MeOH:triethylamine (89:1:1,v/v) furnished the title compound as homogeneous material. Pooling andconcentration of appropriate fractions furnished the 5'-O-DMT nucleosideas a foam (1.5 g, 66.5%).

¹ H NMR (MeSO-d₆) δ 2.02 (3H, s, SCH₃), 3.15-3.55 (1H, m, 2'CH), 3.75(6H, s, 2 OCH₃), 3.97 (1H, m, 4'H), 4.24 (1H, m, 3'H), 5.48 (1H, d, C₅H), 5.73 (1H, d, 3'-OH), 6.03 (1H, d, C1'H), 6.82-7.4 (13H, m, ArH),6.65 (1H, d, C₆ H), 11.4 (1H, br s, NH).

G. 2'-Deoxy-3'-O-(N,N-diisopropyl)-O-β-cyanoethylphosphoramide!-5'-O-(4,4'-dimethoxytrityl)-2'-Methylthiouridine

To a stirred solution of2'-deoxy-5'-O-(4,4'-dimethoxytrityl)-2'-methylthiouridine (1.5 g, 2.67mmol) in dry THF (25 ml) was added diisopropylethylamine (1.4 ml, 8mmol) and the solution cooled to 0° C.N,N-diisopropyl-β-cyanoethylphosphoramidic chloride (1.26 ml, 5.34 mmol)was added dropwise over a period of 15 min. The reaction mixture wasthen stirred at room temperature for 2 hr. EtOAc (100 ml, containing 1%triethylamine) was added and the solution washed with sat NaCl (2×50 ml)and the organic layer dried over MgSO₄. The solvent was removed underpressure and the residue purified by short silica gel (30 g) columnchromatography. Elution with CH₂ Cl₂ :MeOH:triethylamine (98:1:1, v/v)furnished the product as a mixture of diastereoisomers. Evaporation ofthe appropriate fractions provided the title compound as a foam (1.32 g,64.7%).

¹ H NMR (CDCl₃) δ 2.0 and 2.02 (3H, 2s, SCH₃), 5.3 and 5.35 (1H, 2d, C₅H), 6.23 (1H, d, 1'M), 7.8 and 7.78 (1H, 2d, C₆ H) and other protons. ³¹P NMR (CDCl₃) δ 151.68 and 152.2 ppm.

H. 2'-Deoxy-3',5'-di-O-Acetyl-2'-Methylthiouridine

2'-Deoxy-2'-methylthiouridine (5.0 g, 18.24 mmol) and acetic anhydride(5.6 ml, 54.74 mmol) were stirred together in dry pyridine (30 ml) atroom temperature for 12 hr. The products were then concentrated underreduced pressure and the residue obtained was purified by short silicagel column chromatography. The appropriate fractions, which were elutedwith CH₂ Cl₂ :MeOH (9:1, v/v), were combined, evaporated under reducedpressure and the residue was crystallized from EtOH to give the titlecompound (6.0 g, 91.8%) as white needles, m.p. 132° C.

¹ H NMR (CDCl₃) δ 2.17 (3H, s, SCH₃), 2.20 (6H, s, 2 COCH₃), 3.40 (1H,t, 2'H), 4.31-4.40 (3H, m, 4',5'H), 5.31 (1H, m, 3'H), 5.80 (1H, d, C₅H), 6.11 (1H, d, 1'H), 7.45 (1H, d, C₆ H), 8.7 (1H, br s, NH).

I.2'-Deoxy-3',5'-di-O-Acetyl-4-(1,2,4-triazol-1-yl)-2'-Methylthiouridine

Triethylamine (8.4 ml, 60.3 mmol) and phosphoryl chloride (1.2 ml, 12.9mmol) were added to a stirred solution of2'-deoxy-3',5'-di-O-acetyl-2'-methylthiouridine (4.6 g, 13 mmol) in CH₃CN (50 ml). 1,2,4-Triazole (4.14 g, 59.9 mmol) was then added and thereactants were stirred together at room temperature. After 16 hr,triethylamine-water (6:1, v/v; 20 ml) followed by sat. aq. NaHCO₃ (100ml) were added to the products and the resulting mixture was extractedwith CH₂ Cl₂ (2×100 ml). The organic layer was dried (MgSO₄) andevaporated under reduced pressure. The residue was purified by shortsilica gel column chromatography. The appropriate fractions, which wereeluted with CH₂ Cl₂ :MeOH (9:1, v/v), were evaporated under vacuum andthe residue was crystallized from EtOH to give the title compound (3.01g, 56.4%) as pale needles, m.p. 127°-130° C.

¹ H NMR (CDCl₃) δ 2.18 (6H, s, 2 COCH₃), 2.30 (3H, s, SCH₃), 3.67 (1H,m, 2'H), 4.38-4.50 (3H, m, 4',5'H), 5.17 (1H, t, 3'H), 6.21 (1H, d,1'H), 7.08 (1H, d, C₅ H), 8.16 (1H,s, CH), 8.33 (1H, d, C₆ H), 9.25 (1H,s, CH).

J. 2'-Deoxy-2'-Methylthiocytidine

2'-Deoxy-3',5'-di-O-acetyl-4-(1,2,4-triazol-1-yl)-2-methylthiouridine(3.0 g, 7.5 mmol) was dissolved in a saturated solution of ammonia inMeOH (70 ml) and the solution was stirred at room temperature in apressure bottle for 3 days. The products were then concentrated underreduced pressure and the residue was crystallized from EtOH:CH₂ Cl₂ togive the title compound (1.06 g, 51.7%) as crystals, m.p. 201° C.

¹ H NMR (Me₂ SO-d₆) δ 1.95 (3H, s, SCH₃), 3.36 (1H, m, 2'H), 3.55 (2H,m, 5'CH₂), 3.82 (1H, m, 4'H), 4.18 (1H, dd, 3'H), 5.75 (1H, d, C₅ H),6.1 (1H, d, 1'H), 7.77 (1H, d, C₆ H).

Anal. calcd. for C₁₀ H₁₅ N₃ O₄ S: C, 43.94; H, 5.53; N, 15.37; S, 11.73.Found, C, 44.07; H, 5.45; N, 15.47; S, 11.80.

K. 2'-Deoxy-N⁴ -Benzoyl-2'-Methylthiocytidine

To a stirred solution of 2'-deoxy-2'-methylthiocytidine (0.86 g, 3.15mmol) in dry pyridine (20 ml) was added trimethylchlorosilane (2.0 ml,15.75 mmol), and stirring continued for 15 min. Benzoyl chloride (2.18ml, 18.9 mmol) was added to the solution followed by stirring for 2 hr.The mixture was then cooled in an ice-bath and MeOH (10 ml) was added.After 5 mins., NH₄ OH (20 ml, 30% aq.) was added and the mixture stirredfor 30 min. The reaction mixture was then concentrated under vacuum andthe residue purified by short silica gel (70 g) column chromatography.Elution with CH₂ Cl₂ :MeOH (9:1, v/v), pooling of appropriate fractionsand evaporation furnished the title compound (0.55 g, 46.6%) whichcrystallized from EtOH as needles, m.p. 193°-194° C.

L. N⁴ -Benzoylamino-1-2-Deoxy-5-(4,4'-Dimethoxytrityl)-2-Methylthio-β-D-Ribofuranosyl!pyrimidin-3(2H)-one(or 2'-Deoxy-N⁴-Benzoyl-5'-(4,4'-Dimethoxytrityl)-2'-Methylthiocytidine)

To a stirred solution of 2'-deoxy-N⁴ -benzoyl-2'-methylthiocytidine(0.80 g, 2.12 mmol) in dry pyridine (10 ml) was added4,4'-dimethoxytrityl chloride (1.16 g, 3.41 mmol) and4-dimethylaminopyridine (10 mg) at room temperature. The solution wasstirred for 2 hr and the products concentrated under vacuum. The residuewas dissolved in CH₂ Cl₂ (70 ml), washed with sat. NaHCO₃ (50 ml), sat.NaCl (2×50 ml), dried (MgSO₄) and evaporated under reduced pressure. Theresidue was purified by short silica gel (50 g) column chromatography.Elution with CH₂ Cl₂ :triethylamine (99:1, v/v), pooling andconcentration of appropriate fractions furnished the title compound(1.29 g, 90%) as a White foam.

¹ H NMR (DMSO-d₆) δ 2.1 (3H, s, SCH₃), 3.5 (1H, m, 2'H), 3.75 (6H, s,OCH₃), 4.15 (1H, m, 4'H) 4.4 (1H, t, 3'H), 5.74 (1H, br d, 3'OH), 6.15(1H, d, C1'H) 6.8-8.0 (25H, m, ArH, and C₅ H), 8.24 (1H, d, C₆ H), 11.3(1H, br s, NH).

M. 2'-Deoxy-N⁴ -Benzoyl-3-O-(N,N-Diisopropyl)-β-Cyanoethylphosphoramide!-5'-O'(4,4'-Dimethoxytrityl)-2'-Methylthiocytidine

2'-Deoxy-N⁴ -benzoyl-5'-(4,4'-dimethoxytrityl)-2'-methylthiocytidine(1.41 g, 2.07 mmol) was treated with diisopropylethylamine (1.4 ml, 8mmol) and N,N-diisopropyl-β-cyanoethylphophoramide chloride (1.26 ml,5.34 mmol) in dry THF (25 ml) as described in Example 8-G above. Thecrude product was purified by short silica gel (50 g) chromatography tofurnish the title compound on elution with CH₂ Cl₂:hexanes:triethylamine (89:10:1, v/v). The appropriate fractions weremixed and evaporated under pressure to give the title compound (1.30 g,71%) as a white foam (mixture of diastereoisomers).

¹ H NMR (CDCl₃) δ 2.31 (3H, s, SCH₃), 3.45-3.7 (3H, m, 2'H and 5'CH₂),3.83 (6H, s, OCH₃), 4.27-4.35 (1H, m, 4'H), 4.6-4.8 (1H, m, 3'H), 6.35(1H, 2d, 1'H), 6.82-7.8 (25H, m, ArH and C₅ H), 8.38 and 8.45 (1H, 2d,C₆ H) and other protons. ³¹ P NMR δ 151.03 and 151.08 ppm.

N. 2'-Deoxy-2'-Methylsulfinylcytidine

2'-Deoxy-2'-methylthiocytidine of Example 8-J was treated as per theprocedure of Example 8-D to yield the title compound as a mixture ofdiastereoisomers having a complex ¹ H NMR spectrum.

O. 2'-Deoxy-2'-Methylsulfonylcytidine

2'-Deoxy-2'-methylthiocytidine of Example 8-J was treated as per theprocedure of Example 8-E to yield the title compound.

P. N⁶ -Benzoyl-3',5'-di-O-Tetrahydropyran-2-yl!-2'-Deoxy-2'-Methylthioadenosine

N⁶ -Benzoyl-9-2'-O-trifluoromethylsulfonyl-3',5'-di-O-(tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adeninefrom Example 1-D is prepared by treatment with methanethiol in thepresence of tetramethylguanidine to yield the title compound.

Q. N⁶ -Benzoyl-2'-Deoxy-2'-Methylthioadenosine

N⁶-Benzoyl-3',5'-di-O-(tetrahydropyran-2-yl)-2'-deoxy-2'-methylthioadenosinefrom Example 8-P is treated as per Example 1-F to yield the titlecompound.

R. N⁶ -Benzoyl-2'-Deoxy-2'-Methylsulfinyladenosine

N⁶ -Benzoyl-2'-deoxy-2'-methylthioadenosine from Example 8-Q was treatedas per the procedure of Example 8-D to yield the title compound.

S. N⁶ -Benzoyl-2'-Deoxy-2'-Methylsulfonyladenosine

N⁶ -Benzoyl-2'-deoxy-2'-methylthioadenosine from Example 8-Q was treatedas per the procedure of Example 8-E to yield the title compound.

T. N²-Isobutyryl-3',5'-di-O-(tetrahydropyran-2-yl)-2'-Deoxy-2'-Methylthioguanosine

N² -Isobutyryl-9-(3',5'-di-O-tetrahydropyran-2-yl!-2'-O-trifluoromethylsulfonyl-β-D-arabinofuranosyl)guaninefrom Example 1-P is treated with methanethiol in the presence of1,1,3,3-tetramethylguanidine to yield the title compound.

U. N² -Isobutyryl-2'-Deoxy-2'-Methylthioguanosine

N²-Isobutyryl-3',5'-di-O-(tetrahydropyran-2-yl)-2'-deoxy-2'-methylthioguanosineis treated as per Example 1-R to yield the title compound.

V. N² -Isobutyryl-2'-Deoxy-2'-Methylsulfinylguanosine

N² -Isobutyryl-2'-Deoxy-2'-methylthioguanosine from Example 8-U wastreated as per the procedure of Example 8-D to yield the title compound.

W. N² -Isobutyryl-2'-Deoxy-2'-Methylsulfonylguanosine

N² -Isobutyryl-2'-Deoxy-2'-methylthioguanosine from Example 8-U wastreated as per the procedure of Example 8-E to yield the title compound.

X. 2'-Deoxy-5-O-(4,4'-Dimethoxytrityl)-2-Methylsulfinyluridine

2'-Deoxy-2'-methylsulfinyluridine from Example 8-D above is treated asper Example 8-F to yield the title compound.

Y. 2'-Deoxy-3'-O-(N,N-Diisopropyl)-O-β-cyanoethylphosphoramide!-5'-O-(4,4'-Dimethoxytrityl)-2'-Methylsulfinyluridine

2'-Deoxy-5'-O-(4,4'-dimethoxytrityl)-2-methylsulfinyluridine is treatedas per Example 8-G to yield the title compound.

Z. N⁶-Benzoyl-2'-Deoxy-5-O-(4,4'-Dimethoxytrityl)-2'-Methylthioadenosine

N⁶ -benzoyl-2'-Deoxy-2'-methylthioadenosine from Example 8-Q above istreated as per Example 8-F to yield the title compound.

AA. N⁶ -Benzoyl-2'-Deoxy-3'-O-(N,N-Diisopropyl)-O-β-Cyanoethylphosphoramide!-5'-O-(4,4'-Dimethoxytrityl)-2'-Methylthioadenosine

N⁶ -benzoyl-2'-Deoxy-5'-O-(4,4'-dimethoxytrityl)-2'-methylthioadenosineis treated as per Example 8-G to yield the title compound.

BB. 2'-Deoxy-N²-Isobutyryl-5-O-(4,4'-Dimethoxytrityl)-2'-Methylthioguanosine

2'-Deoxy-N² -isobutyryl-2'-methylthioguanosine from Example 8-U above istreated as per Example 8-F to yield the title compound.

CC. 2'-Deoxy-N² -Isobutyryl-3'-O-(N,N-Diisopropyl)-O-β-Cyanoethylphosphoramide!-5'-O-(4,4'-Dimethoxytrityl)-2'-Methylthioguanosine

2'-Deoxy-N²-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-methylthioguanosine istreated as per Example 8-G to yield the title compound.

DD. 2'-Deoxy-5-O-(4,4'-Dimethoxytrityl)-2'-Methylsulfonyluridine

2'-Deoxy-2'-methylsulfonyluridine from Example 8-E above is treated asper Example 8-F to yield the title compound.

EE. 2'-Deoxy-3'-O-(N,N-Diisopropyl)-O-β-Cyanoethylphosphoramide!-5'-O-(4,4'-Dimethoxytrityl)-2'-Methylsulfinyluridine

2'-Deoxy-5'-O-(4,4'-dimethoxytrityl)-2'-methylsulfinyluridine is treatedas per Example 8-G to yield the title compound.

EXAMPLE 9

Chemical conversion of an thymine or cytosine (pyrimidine type base) toits β-D-2'-deoxy-2'-substituted erythro-pentofuranosyl nucleoside;2'-substituted ribosylation).

The thymine or cytosine type analogs are trimethylsilylated understandard conditions such as hexamethyldisilazane (HMDS) and an acidcatalyst (ie. ammonium chloride) and then treated with3,5-O-ditoluoyl-2-deoxy-2-substituted-α-D-erythro-pentofuranosylchloride in the presence of Lewis acid catalysts (ie. stannic chloride,iodine, boron tetrafluoroborate, etc.). A specific procedure hasrecently been described by J. N. Freskos, Nucleosides & Nucleotides8:1075-1076 (1989) in which copper (I) iodide is the catalyst employed.

EXAMPLE 10

Chemical conversion of an adenine or guanine (purine type base) to itsβ-D-2'-deoxy-2'-substituted erythro-pentofuranosyl nucleoside;2'-substituted ribosylation).

The protected purine type analogs are converted to their sodium saltsvia sodium hydride in acetonitrile and are then treated with3,5-O-ditoluoyl-2-deoxy-2-substituted-α-D-erythro-pentofuranosylchloride at ambient temperature. A specific procedure has recently beendescribed by R. K. Robins et al., Journal of American Chemical Society106:6379 (1984).

EXAMPLE 11

Conversion of 2'-deoxy-2-substituted thymidines to the corresponding2'-deoxy-2'-substituted cytidines (chemical conversion of an pyrimidinetype 4-keto group to an 4-amino group).

The 3',5'-sugar hydroxyls of the 2'-modified nucleoside types areprotected by acyl groups such as toluoyl, benzoyl, p-nitrobenzoyl,acetyl, isobutryl, trifluoroacetyl, etc. using standards conditions ofthe acid chlorides or anhydrides and pyridine/dimethylaminopyridinesolvent and catalyst. The protected nucleoside is now chlorionated withthionyl chloride or phosphoryl chloride in pyridine or other appropriatebasic solvents. The pyrimidine type 4-chloro groups or now displacedwith ammonium in methanol. Deprotection of the sugar hydroxyls alsotakes place. The amino group is benzoylated by the standard two-stepprocess of complete benzylation (sugar hydroxyls and amino group) andthe acyls are selectively removed by aqueous sodium hydroxide solution.Alternatively, the in situ process of first treating the nucleoside withchlorotrimethylsilane and base to protect the sugar hydroxyls fromsubsequent acylation may be employed. K. K. Ogilvie, Can J. Chem.67:831-839 (1989). Another conversion approach is to replace thepyrimidine type 4-chloro group with an 1,2,4-triazolo group whichremains intact throughout the oligonucleotide synthesis on the DNAsynthesizer and is displaced by ammonium during the ammonium hydroxidestep which removes the oligonucleotide from the CPG support anddeprotection of the heterocycles. Furthermore, in many cases thepyrimidine type 4-chloro group can utilized as just described andreplaced at the end of the oligonucleotide synthesis.

EXAMPLE 12

Procedure for the attachment of 2'-deoxy-2'-substituted5'-dimethoxytriphenylmethyl ribonucleosides to the 5'-hydroxyl ofnucleosides bound to CPG support.

The 2'-deoxy-2'-substituted nucleosides that will reside in the terminal3'-position of certain antisense oligonucleotides is protected as their5'-DMT (the cytosine and adenine exocyclic amino groups are benzoylatedand the guanine amino is isobutyrylated) and treated withtrifluoro-acetic acid/bromoacetic acid mixed anhydride in pyridine anddimethylaminopyridine at 50° C. for five hours. The solution isevaporated under reduced pressure to a thin syrup which is dissolved inethyl acetate and passed through a column of silica gel. The homogenousfractions were collected and evaporated to dryness. A solution of 10 mlof acetonitrile, 10 micromoles of the 3'-O-bromomethylester modifiedpyrimidine nucleoside, and one ml of pyridine/dimethylaminopyridine(1:1) is syringed slowly (60 to 90 sec) through a one micromole columnof CPG thymidine (Applied Biosystems, INC.) that had previously beentreated with acid according to standard conditions to afford the free5'-hydroxyl group. Other nucleoside bound CPG columns could be employed.The eluent is collected and syringed again through the column. Thisprocess is repeated three times. The CPG column is washed slowly with 10ml of acetonitrile and then attached to an ABI 380B nucleic acidsynthesizer. Oligonucleotide synthesis is now initiated. The standardconditions of concentrated ammonium hydroxide deprotection that cleavesthe thymidine ester linkage from the CPG support also cleaves the 3',5'ester linkage connecting the pyrimidine modified nucleoside to thethymidine that was initially bound to the CPG nucleoside. In thismanner, any 2'-substituted nucleoside or generally any nucleoside withmodifications in the heterocycle and/or sugar can be attached at thevery 3'-end of an oligonucleotide sequence.

EXAMPLE 13

Procedure for the conversion of 2'-deoxy-2'-substitutedribonucleoside-5'-DMT-3'-phosphoramidites into oligonucleotides.

The polyribonucleotide solid phase synthesis procedure of B. S. Sproat,et al., Nucleic Acids Research 17:3373-3386 (1989) is utilized toprepare the 2'-modified oligonucleotides.

Oligonucleotides of the sequence CGA CTA TGC AAG TAC having2'-deoxy-2'-fluoro substituent nucleotides were incorporated at variouspositions within this sequence. In a first oligonucleotide each of theadenosine nucleotides at positions 3, 6, 10, 11 and 14 (counted in a 5'to 3' directed) were modified to include a 2'-deoxy-2'-fluoro moiety. Ina further oligonucleotide, the adenosine and the uridine nucleotides atpositions 3, 5, 6, 7, 10, 11, 13 and 14 were so modified. In even afurther oligonucleotide, the adenosine, uridine and cytidine nucleotidesat positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 13 and 14 were so modified andin even a further oligonucleotide, the nucleotides (adenosine, uridine,cytidine and guanosine) at every position was so modified. Additionallyan oligonucleotide having the sequence CTC GTA CCT TCC GGT CC wasprepared having adenosine, uridine and cytidine nucleotides at positions1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 15 and 16 also modified to contain2'-deoxy-2'-fluoro substituent moieties.

Various oligonucleotides were prepared that incorporated nucleotideshaving 2'-deoxy-2'-methylthio substituents. For ascertaining thecoupling efficiencies of 2'-deoxy-2'-methylthio bearing nucleotides intooligonucleotides, the trimer TCC and the tetramer TUU U weresynthesized. In the trimer, TCC, the central cytidine nucleotide (thesecond nucleotide) included a 2'-deoxy-2'-methylthio substituent. In thetetramer, each of the Uridine nucleotides included a2'-deoxy-2'-methylthio substituent. In further oligonucleotides,2'-deoxy-2'-methylthio substituent bearing nucleotides were incorporatedwithin the oligonucleotide sequence in selected sequence positions. Eachof the nucleotides at the remaining sequence positions incorporated a2'-O-methyl substituent on its nucleotide. Thus all of the nucleotideswithin the oligonucleotide included a substituent group thereon, eithera 2'-deoxy-2'-methylthio substituent or a 2'-O-methyl substituent. Theseoligonucleotides are: GAG CUC CCA GGC having 2'-deoxy-2'-methylthiosubstituents at positions 4, 5, 6, 7 and 8; CGA CUA UGC AAG UAC having2'-deoxy-2'-methylthio substituents at positions 1, 4, 5, 7, 9 and 13;UCC AGG UGU CCG AUC having 2'-deoxy-2'-methylthio substituents arepositions 1, 2, 3, 7, 9, 10, 11 and 14; TCC AGG CCGUUU C having2'-deoxy-2'-methylthio substituents at positions 10, 11 and 12; and TCCAGG TGT CCC C having 2'-deoxy-2'-methylthio substituents at positions10, 11 and 12.

EXAMPLE 14

Preparation of 2'-Deoxy-2'-fluoro Modified PhosphorothioatesOligonucleotides.

2'-Deoxy-2'-substituted 5'-DMT nucleoside 3'-phosphoramidites preparedas described in Examples 1-7 were inserted into sequence-specificoligonucleotide phosphorothioates as described by S. Beaucage et al.,Journal of American Chemical Society 112:1253-1255 (1990) and B. S.Sproat, et al., Nucleic Acids Research 17:3373-3386 (1989).

Oligonucleotides of the sequence CGA CTA TGC AAG TAC havingphosphorothioate backbone linkages and 2'-deoxy-2'-fluoro substituentnucleotides were incorporated at various positions within this sequence.In a first oligonucleotide each of the backbone linkages was aphosphorothioate linkage and each of the adenosine, uridine and cytidinenucleotides at positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 13 and 14 (countedin a 5' to 3' directed) were modified to include a 2'-deoxy-2'-fluoromoiety. In a further oligonucleotide each of the backbone linkages was aphosphorothioate linkages and the nucleotides (adenosine, uridine,cytidine and guanosine) at every position were modified to include a2'-deoxy-2'-fluoro moiety.

EXAMPLE 15

Preparation of 2'-Deoxy-2'-fluoro Modified Phosphate MethylatedOligonucleotides.

The protection, tosyl chloride mediated methanolysis, and milddeprotection described by L. H. Koole et al., in the Journal of OrganicChemistry 54:1657-1664 (1989), is applied to 2'-substitutedoligonucleotides to afford phosphate-methylated 2'-substitutedoligonucleotides.

EXAMPLE 16

Hybridization Analysis.

A. Evaluation of the thermodynamics of hybridization of 2'-modifiedoligonucleotides.

The ability of the 2'-modified oligonucleotides to hybridize to theircomplementary RNA or DNA sequences was determined by thermal meltinganalysis. The RNA complement was synthesized from T7 RNA polymerase anda template-promoter of DNA synthesized with an Applied Biosystems, Inc.380B RNA species was purified by ion exchange using FPLC (LKBPharmacia,Inc.). Natural antisense oligonucleotides or those containing2'-modifications at specific locations were added to either the RNA orDNA complement at stoichiometric concentrations and the absorbance (260nm) hyperchromicity upon duplex to random coil transition was monitoredusing a Gilford Response II spectrophotometer. These measurements wereperformed in a buffer of 10 mM Na-phosphate, pH 7.4, 0.1 mM EDTA, andNaCl to yield an ionic strength of 10 either 0.1M or 1.0M. Data wasanalyzed by a graphic representation of 1/T_(m) vs ln Ct!, where Ct! wasthe total oligonucleotide concentration. From this analysis thethermodynamic para-meters were determined. Based upon the informationgained concerning the stability of the duplex of heteroduplex formed,the placement of nucleotides containing 2'-deoxy-2'-substituents intooligonucleotides were assessed for their effects on helix stability.Modifications that drastically alter the stability of the hybrid exhibitreductions in the free energy (delta G) and decisions concerning theirusefulness as antisense oligonucleotides were made.

As is shown in the following Table 1, the incorporation of2'-deoxy-2'-fluoro nucleotides into oligonucleotides resulted insignificant increases in the duplex stability of the modifiedoligonucleotide strand (the antisense strand) and its complementary RNAstrand (the sense strand). In both phosphodiester backbone andphosphorothioate backbone oligonucleotides, the stability of the duplexincreased as the number of 2'-deoxy-2'-fluoro containing nucleotides inthe antisense strand increased. As is evident from Table 1, withoutexception, the addition of a 2'-deoxy-2'-fluoro bearing nucleotide,irrespective of the individual substituent bearing nucleotide orirrespective of the position of that nucleotide in the oligonucleotidesequence, resulted in a increase in the duplex stability.

In Table 1, the underline nucleotides represent nucleotides that includea 2'-deoxy-2'-fluoro substituent. The non-underlined nucleotides arenormal nucleotides. The oligonucleotides prefaced with the designation"ps" have a phosphorothioate backbone. Unlabeled oligonucleotides arenormal phosphodiester backboned oligonucleotides.

                                      TABLE 1                                     __________________________________________________________________________    EFFECTS OF 2'-DEOXY-2'-FLUORO MODIFICATIONS ON DNA(ANTISENSE)                 RNA(SENSE) DUPLEX STABILITY                                                                 G°37                                                                          G°37    Tm(°(C.)/                          Antisence Sequence                                                                          (kcal/mol)                                                                           (kcal/mol)                                                                           Tm(°C.)                                                                    Tm(°C.)                                                                    subst                                     __________________________________________________________________________    CGA CTA TGC AAG TAC                                                                         -10.11 ± 0.04                                                                            45.1                                              CG A CT A TGC -13.61 ± 0.08                                                                     -3.50 ± 0.09                                                                      53.0                                                                              +7.9                                                                              +1.6                                      CG A C UA  UG C  AAG  UAC                                                                   -16.18 ± 0.08                                                                     -6.07 ± 0.09                                                                      58.9                                                                              +13.8                                                                             +1.7                                       CG A  CUA  UG C  AAG  UAC                                                                  -19.85 ± 0.05                                                                     -9.74 ± 0.06                                                                      65.2                                                                              +20.1                                                                             +1.8                                      ps(CGA CTA TGC AAG TAC)                                                                      -7.58 ± 0.06                                                                            33.9                                                                              -11.2                                         ps( CG A  CUA -15.90 ± 0.34                                                                     -8.32 ± 0.34                                                                      60.9                                                                              27.0                                                                              +2.5                                      CTC GTA CCT TCC GGT CC                                                                      -14.57 ± 0.13                                                                            61.6                                               CUC G UA  CCU  UCC GG U  CC                                                                -27.81 ± 0.05                                                                     -13.24 ± 0.14                                                                     81.6                                                                              +20.0                                                                             +1.4                                      __________________________________________________________________________

As is evident from Table 1, the duplexes formed between RNA and anoligonucleotides containing 2'-deoxy-2'-fluoro substituted nucleotidesexhibited increased binding stability as measured by the hybridizationthermodynamic stability. Delta Tm's of greater than 20° C. weremeasured. By modifying the backbone to a phosphorothioate backbone evengreater delta Tm's were observed. In this instance delta Tm's greaterthan 31° C. were measured. These fluoro substituted oligonucleotidesexhibited a consistent and additive increase in the thermodynamicstability of the duplexes formed with RNA. While we do not wish to bebound by theory, it is presently believed that the presence of the2'-fluoro substituent results in the sugar moiety of 2'-fluorosubstituted nucleotide assuming substantially a 3'-endo conformation andthis results in the oligonucleotide-RNA duplex assuming an A-typehelical conformation.

B. Fidelity of hybridization of 2'-modified oligonucleotides

The ability of the 2'-modified antisense oligonucleotides to hybridizewith absolute specificity to the targeted mRNA was shown by Northernblot analysis of purified target mRNA in the presence of total cellularRNA. Target mRNA was synthesized from a vector containing the cDNA forthe target mRNA located downstream from a T7 RNA polymerase promoter.Synthesized mRNA was electrophoresed in an agarose gel and transferredto a suitable support membrane (ie. nitrocellulose). The supportmembrane was blocked and probed using ³² P!-labeled antisenseoligonucleotides. The stringency was determined by replicate blots andwashing in either elevated temperatures or decreased ionic strength ofthe wash buffer. Autoradiography was performed to assess the presence ofheteroduplex formation and the autoradiogram quantitated by laserdensitometry (LKB Pharmacia, Inc.). The specificity of hybrid formationwas determined by isolation of total cellular RNA by standard techniquesand its analysis by agarose electrophoresis, membrane transfer andprobing with the labeled 2'-modified oligonucleotides. Stringency waspredetermined for the unmodified antisense oligonucleotides and theconditions used such that only the specifically targeted mRNA wascapable of forming a heteroduplex with the 2'-modified oligonucleotide.

C. Base-Pair Specificity of Oligonucleotides and RNA

Base-pair specificity of 2-deoxy-2'-fluoro modified oligonucleotideswith the RNA complement (a "Y strand") was determined by effectingsingle base-pair mismatches and a bulge. The results of thesedeterminations are shown in Table 2. An 18 mer "X strand"oligonucleotide containing 14 adenosine, uridine and cytidinenucleotides having a 2'-deoxy-2'-fluoro substituent was hybridized withthe RNA complement "Y strand" in which the 10 position was varied. InTable 2, the underline nucleotides represent nucleotides that include a2'-deoxy-2'-fluoro substituent.

                                      TABLE 2                                     __________________________________________________________________________    EFFECTS OF SINGLE BASE MISMATCHES ON 2'-DEOXY-2'-FLUORO                       MODIFIED DNA · RNA DUPLEX STABILITY                                      base-pair                                                                             G°37                                                                            G°37                                              Y   type    (kcalol) (kcal/mol)                                                                             Tm(°C.)                                                                     Tm(°C.)                             __________________________________________________________________________    X strand: deoxy(CTC GTA CCT TTC CGG TCC)                                      Y strand: ribo(.sup.3' GAG CAU GGY AAG GCC AGG.sup.5')                        A   Watson-Crick                                                                          -14.57 ± 0.13  61.6                                            C   T--C mismatch                                                                         -12.78 ± 0.11                                                                       1.79 ± 0.17                                                                         54.4 -7.2                                       G   T--G mismatch                                                                         -16.39 ± 0.25                                                                       -1.82 ± 0.28                                                                        61.7 0.1                                        U   T--U mismatch                                                                         -13.48 ± 0.17                                                                       1.09 ± 0.22                                                                         55.9 -5.7                                       none                                                                              bulged T                                                                              -14.86 ± 0.35                                                                       -0.284 ± 0.37                                                                       59.4 -2.2                                       X strand: deoxy( CUC G UA  CCU  UUC  CGG  UCC)                                Y strand: ribo(.sup.3' GAG CAU GGY AAG CCC AGG.sup.5')                        A   Watson-Crick                                                                          -27.80 ± 0.05  81.6                                            C   U--C mismatch                                                                         -21.98 ± 0.26                                                                       5.82 ± 0.28                                                                         73.8 -7.8                                       G   U--G mismatch                                                                         -21.69 ± 0.16                                                                       6.12 ± 0.17                                                                         77.8 -3.8                                       U   U--U mismatch                                                                         -18.68 ± 0.15                                                                       9.13 ± 0.16                                                                         73.6 -8.0                                       none                                                                              bulged U                                                                              -22.87 ± 0.27                                                                       4.94 ± 0.27                                                                         75.5 -6.2                                       __________________________________________________________________________

As is evident from Table 2, the 2'-deoxy-2'-fluoro modifiedoligonucleotide formed a duplex with the RNA complement with greaterspecificity than a like sequenced unmodified oligonucleotide.

EXAMPLE 17

Nuclease Resistance

A. Evaluation of the resistance of 2'-modified oligonucleotides to serumand cytoplasmic nucleases.

Natural phosphorothioate, and 2-modified oligonucleotides were assessedfor their resistance to serum nucleases by incubation of theoligonucleotides in media containing various concentrations of fetalcalf serum or adult human serum. Labeled oligonucleotides were incubatedfor various times, treated with protease K and then analyzed by gelelectrophoresis on 20% polyacrylamine-urea denaturing gels andsubsequent autoradiography. Autoradiograms were quantitated by laserdensitometry. Based upon the location of the modifications and the knownlength of the oligonucleotide it was possible to determine the effect onnuclease degradation by the particular 2'-modification. For thecytoplasmic nucleases, a HL60 cell line was used. A post-mitochondrialsupernatant was prepared by differential centrifugation and the labeledoligonucleotides were incubated in this supernatant for various times.Following the incubation, oligo-nucleotides were assessed fordegradation as outlined above for serum nucleolytic degradation.Autoradiography results were quantitated for comparison of theunmodified, the phosphorothioates, and the 2'-modified oligonucleotides.Utilizing these test systems, the stability of a 15-mer oligonucleotidehaving 2-deoxy-2'-fluoro substituted nucleotides at positions 12 and 14and a phosphorothioate backbone was investigated. As a control, anunsubstituted phosphodiester oligonucleotide was 50% degraded within 1hr and 100% degraded within 20 hours. In comparison for the2'-deoxy-2'-fluoro substituted oligonucleotide having thephosphorothioate backbone, degradation was limited to less than 10%after 20 hours.

B. Evaluation of the resistance of 2'-modified oligonucleotides tospecific endo- and exo-nucleases.

Evaluation of the resistance of natural and 2'-modified oligonucleotidesto specific nucleases (ie, endonucleases, 3',5'-exo-, and5',3'-exonucleases) was done to determine the exact effect of themodifications on degradation. Modified oligonucleotides were incubatedin defined reaction buffers specific for various selected nucleases.Following treatment of the products with proteinase K, urea was addedand analysis on 20% poly-acrylamide gels containing urea was done. Gelproducts were visualized by staining using Stains All (Sigma ChemicalCo.). Laser densitometry was used to quantitate the extend ofdegradation. The effects of the 2'-modifications were determined forspecific nucleases and compared with the results obtained from the serumand cytoplasmic systems.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 7                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15                                                                (B) TYPE: nucleic                                                             (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      CGACTATGCAAGTAC15                                                             (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15                                                                (B) TYPE: nucleic                                                             (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      CGACUAUGCAAGUAC15                                                             (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17                                                                (B) TYPE: nucleic                                                             (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      CTCGTACCTTCCGGTCC17                                                           (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17                                                                (B) TYPE: nucleic                                                             (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      CUCGUACCUUCCGGUCC17                                                           (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic                                                             (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      CTCGTACCTTTCCGGTCC18                                                          (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic                                                             (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      GAGCAUGGYAAGGCCAGG18                                                          (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic                                                             (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      CUCGUACCUUUCCGGUCC18                                                          __________________________________________________________________________

What is claimed is:
 1. An oligonucleotide that hybridizes with RNA orDNA, having 5 to 50 covalently-bound nucleosides that individuallyinclude a ribose or deoxyribose sugar portion and a base portion,wherein:said sugar portions of said nucleosides are joined together by3'-5' internucleoside linkages such that the base portions of saidnucleosides form a mixed base sequence that is complementary to an RNAbase sequence or to a DNA base sequence; and at least two of saidnucleosides include a modified deoxyfuranosyl moiety bearing a 2'-fluorosubstituent; and wherein a duplex formed between said oligonucleotideand its complement exhibits greater thermal stability than does a duplexformed between said complement and an oligonucleotide that does notinclude 2'-fluoro substituents.
 2. The oligonucleotide of claim 1wherein said modified deoxyfuranosyl moiety is a2'-deoxy-2'-fluororibofuranomyl moiety.
 3. The oligonucleotide of claim1 wherein at least two of said nucleosides are covalently bound throughphosphorothioate, methyl phosphonate, or phosphate alkylateinternucleoside linkages.